APMIS 123: 697–705
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12404
Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats ZIXIN MIN,1 WENXIANG ZHAO,1 NANNAN ZHONG,1 YUANXU GUO,1 MENGYAO SUN,1 QUANCHENG WANG,1 RUI ZHANG,1 JIDONG YAN,1 LIFANG TIAN,1 FUJUN ZHANG,1 YAN HAN,1 QILAN NING,1 LIESU MENG,1 JIAN SUN1,2 and SHEMIN LU1,2 1
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an; and 2Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China
Min Z, Zhao W, Zhong N, Guo Y, Sun M, Wang Q, Zhang R, Yan J, Tian L, Zhang F, Han Y, Ning Q, Meng L, Sun J, Lu S. Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats. APMIS 2015; 123: 697–705. This study aimed to observe the effects of Se deficiency on epiphyseal plates of two generation DA rats fed with artificial total synthetic low Se diet. All F0 and F1 DA rats were fed with synthetic low Se diet (SeD group) and low Se diet supplied with Se (SeS group). The levels of selenium and enzyme activities of GPx were detected in plasma of the rats. General growth of bone and articular cartilage was measured macroscopically and microscopically. The epiphyseal plate of femur heads or tibia were obtained to histological and immunohistochemical examinations. The cartilage from left knee joints and femur heads was used to detect the gene expression of collagens, ADAMTSs and several selenoproteins by RT-qPCR. Two generation SeD rats showed Se insufficiency status. The thicknesses of the femur and tibial epiphyseal plates in both F0 and F1 SeD rats were significantly less than that of SeS rats. In F1 generation, SeD rats showed much fewer proliferative chondrocyte layers than SeS ones. Importantly, two generation SeD rats both showed significantly more serious pathological changes of epiphyseal plates. In two generation rats, gene expressions of COL II, GPx1 and GPx4 were significantly down-regulated in SeD rats than SeS ones; meanwhile ADAMTS-4 showed an up-regulated expression in cartilage. Dietary Se deficiency can apparently cause epiphyseal plate lesion and decrease cartilage type II collagen production and GPx1 activity in two generation DA rats fed with the artificial total synthesis low Se diet. Key words: DA rats; selenium deficiency; diet; cartilage lesion. Jian Sun and Shemin Lu, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China. e-mails: [email protected]; [email protected] or [email protected]
Kashin-Beck disease (KBD), an endemic osteoarthropathy in Northern China, is characterized by pathological chondronecrosis of multiple areas of hyaline cartilage including articular cartilage and growth plates from different peripheral joints of the children from endemic areas (1). The disease manifests aberrant metabolism of articular cartilage leading to delayed skeletal development, secondary osteoarthritis and severe disability (2, 3). Concerning etiology of the disease, a combination of different environmental factors has been under considerations, including mycotoxicosis, iodine deficiency and selenium deficiency (4). Selenium (Se) is an essential trace element incorporated into selenoproteins that are associated with Received 20 January 2015. Accepted 17 April 2015
diverse physiological or pathological processes such as development and inflammation in mammals. Intriguingly, osteo-chondroprogenitor-specific deletion of the selenocysteinyl tRNA gene leads to the phenotypes similar to KBD, particularly showing chondronecrosis and abnormal skeletal development in the mice (5). The evidence has documented that Se as well as selenoproteins is important to the development of osteochondral system. Further on, Se supplementation has been used in the prophylatics and therapy of KBD despite of the ambiguous mechanism (6). Several animal models have been used to intuitively mimic the pathological manifestation of Se-deprived cartilage disorder (5). Existing models for KBD include outbred SD or Wistar rats fed with Se deficient or supplementary mycotoxin or 697
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iodine (7, 8). However, the replication of the typical pathological phenotypes of KBD, such as deep chondrocyte necrosis and epiphyseal dysplasia, in the rats by only Se deficiency alone has been proven to be elusive (9). When Wistar rats were used, the same protocol seems to induce an putative osteopenia (10), as the same as in mice (11). Dark Agouti (DA) rats, an inbred strain, are more sensitive to various autoimmune joint diseases and commonly used for investigating the pathogenesis of rheumatoid arthritis (RA). DA rat models include pristane induced arthritis (PIA) (12), adjuvant-oil induced arthritis (13), CXI induced arthritis (CXIIA) and CII induced arthritis (CIIIA) (14, 15). Taking into consideration of much more sensitive to induced arthritis, DA rats were chosen to establish an animal model for KBD, hoping to observe more obvious effects of Se deficiency on cartilage pathology. In this study, we imitated the nutriture of the children from KBD endemic areas, and two generation DA rats were fed with Se deficient diet, to observe effect of Se deficiency on cartilage. We confirmed that Se deficient diet can impact the epiphyseal plate development and induce epiphyseal abnormality, and Se supplementation has a protective role in DA rats. The probable mechanism is related to regulating gene expression of antioxidant and extracellular matrix metabolism relevant molecules in chondrocytes. MATERIALS AND METHODS Rats Dark Agouti rats were bred and maintained in a specific pathogen-free animal house of our department. Age and sex-matched rats were used within all experiments, and each group contained 8 to 10 rats at age of 8 to 12 weeks. The experiments were approved by the Institutional Animal Ethics Committee of the university.
Diets Artificial total synthesis Se deficient and Se sufficient diets were made as R.F. Burk formula (16), and stored in fridge for use of only one week since multi-gelation should be avoided. Se contents were determined to ensure that Se intake from the diets was up to the experimental requirement. The Se sufficient diet was prepared by adding sodium selenite into the Se deficient one.
Animal experiments In F0 generation experiments, 20 DA rats at the age of 8 to 12 weeks were randomly divided into two groups matched by age and sex. Each group had six male and four female rats. The rats in Se sufficient (SeS) group were fed with Se sufficient diet as a control, and the rats in Se deficient (SeD) group were fed with Se deficient diet for 2 months.
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One male F0 rat and 2 to 3 female rats were kept in the same cage and fed with SeS or SeD diet. The produced offsprings would be used in F1 generation experiments. In F1 generation experiments, after weaning the rats were divided into 2 groups fed with SeS and SeD for 2 to 3 months, respectively. In the end of experiments, blood was collected from abdominal aorta to euthanize the rats. And hearts, livers, spleens and kidneys were collected and weighted immediately. All tissues were sampled to fix in the 4% paraformaldehyde for sectioning or to keep in 80 °C for RNA isolation. After removing muscle and other soft tissues, left low limbs were quickly placed in the fixing buffer, while the right femur heads were saved in 80 °C for RNA isolation.
Detection of Se content and GPx activity Contents of Se in diets and sera of the DA rats were determined by fluorometric method fluorescent atomic absorption spectrometry. Total plasma glutathione peroxidase (GPx) activity was measured by using the direct DTNB method.
Histology staining Removed livers, hearts, spleens and kidneys from each rat were fixed in 4% paraformaldehyde in PBS solution and embedded together in paraffin. Knee joints fixed with 4% paraformaldehyde (17) were tenderly decalcified in 10% EDTA liquid for 4 week, subsequently paraffin-embedded for staining to observe morphological changes of epiphyseal plate. Then all sections were cut at 5 lm thickness and stained with hematoxylin and eosin (H&E), otherwise, safranin O and fast green.
Measurement of growth plate and chondrocytes The thickness and chondrocyte layers of epiphyseal plate were measured under light microscope. The images of each epiphyseal plate on sections were obtained under the microscope (Olympus BX51; Olympus, Tokyo, Japan) equipped with a digital camera (Olympus PD71; Olympus). All images should cover the whole structure of epiphyseal plates after merging. Five sites evenly locating in each epiphyseal plate were selected to measure thickness and chondrocyte layers and the average value was obtained to stand for each animal. These values were all calculated by Image-Pro Plus 6 (Media Cybernetics, Silver Spring, MD, USA). Also, femur diameters, femur lengths and tibial lengths were measured by vernier caliper.
Assessment of epiphyseal plate lesion Morphological changes of epiphyseal plate were observed and the abnormal lesions can be divided into three types: column necrosis or missing is defined as a necrosis or only chondrocyte lacuna in a whole column chondrocytes from proliferation layer to the hypertrophy layer; laminar necrosis or missing is defined as necrosis or chondrocyte lacuna in one or a few layers of continuous several columns (at least 2); and chondrocyte cluster is defined as round or elliptical or irregular round cell cluster without adjacent chondrocyte column and lacking at least 2 layer of cells between the upper and the nether layer. © 2015 APMIS. Published by John Wiley & Sons Ltd
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Total RNA extraction Total RNA was isolated from femur head cartilage or internal organ samples using the TRIzolÒ method. Two microgram of each total RNA from cartilage was timely reversed to cDNA according to the manufacturer’s instruction (RevertAidTM; Fermentas Life Sciences, Burlington, ON, Canada) and stored at 20 °C until used.
Real-time quantitative PCR analysis
gene expression assay analysis were calculated by using Student’s T test. Correlation analysis was performed by Spearman test. In all tests a value of p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001). All analyses were performed using the software program GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
RESULTS
The mRNA expressions were tested by Real-time quantitative PCR (RT-qPCR), which was performed on iQ5 realtime PCR detection system (Bio-Rad, Hercules, CA, USA) with SYBRÒ Premix Ex TaqTM II (TaKaRa, Shiga, Japan). The relative gene expression is normalized by GAPDH. The information of primers is depicted in Table S1.
Immunohistochemistry staining Knee joints fixed with 4% paraformaldehyde (17) were decalcified in 10% EDTA liquid for 4 week, subsequently paraffin-embedded and dissected at 5 lm thickness. After intrinsic peroxidase activity was blocked with 3% hydrogen peroxide, the sections were incubated with 1.5% BSA for 1 h. Then sections were covered with 1:200 dilution of polyclonal antibody against type II collagen a1 (COL II a1) and 1:250 monoclonal antibody against GPx1 respectively, for 14 h at 4 °C wet box in fridge. The sections were rinsed and incubated sequentially with biotinylated secondary antibody for 1 h and ABC reagent (Vector Laboratories, Burlingame, CA, USA) for 10 min at room temperature. Rabbit IgG was used as a negative control.
Statistical analysis Data were represented as means SEM. The pathological data were calculated using Mann–Whitney U test, and
Artificial total synthetic Se deficient diet can cause Se insufficiency status of rats
As expected, the Se content in Se deficient synthetic diet was significantly lower than that in Se supplemental one (Table 1). This result confirmed that the diet with Se deficiency can be used for subsequent experiments. The plasma Se contents showed much lower in SeD rats than SeS ones (Table 1). Plasma total GPx activity of the SeD rats showed a significant lower level than the SeS group rats in both F0 and F1 generations, respectively (Fig. 1A). The expressions of liver GPx1 and SEPP1 in the SeD group were both significantly lower than the SeS one (Fig. 1B,C). All these indicated that the rats fed with SeD diet existed in a Se deficient status. Se deficiency can cause epiphyseal plate lesion
As an index of cartilage development, the thickness of tibial and femur epiphyseal plates were overall measured after safranin O and fast green staining (Fig. 2A). In F0 generation, the SeD rats showed a
Table 1. Selenium contents in rat diet and blood samples of each group [mean SEM (n)] Groups Se content in diet (lg/g) Se content in blood (lg/mL) F0 rats F1 rats Se sufficient 0.288 0.038 (10) 0.676 0.044 (10) 0.520 0.054 (8) Se deficient 0.018 0.005 (10)*** 0.262 0.026 (10)*** 0.032 0.009 (6)*** ***p < 0.001 when compared two groups. Se levels were measured by fluorescent atomic absorption spectrometry.
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Fig. 1. Evaluation of Se status in experimental rats. (A) Plasma total GPx activity in each group. The data were expressed as means SEM, * and ** stand for p < 0.05 and 0.01 respectively between two groups (F0 group: n = 10 for each group; F1 group: n = 8 for SeS group, and n = 6 for SeD group). (B) and (C) mRNA expression of GPx1 and SEPP1 in rat livers from F1 group. The data were expressed as means SEM, * stands for p < 0.05 between two groups (F1 rats: n = 8 for SeS group, and n = 6 for SeD group). © 2015 APMIS. Published by John Wiley & Sons Ltd
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Fig. 2. Comparisons of bone and cartilage growth in F0 and F1 rats among 4 groups. (A) Representative images of tibial epiphyseal plates from 4 experimental groups. The sections of rat knee joints were stained with Safranine O and fast-green dye (bar: 200 lm). (B)The thicknesses of femur and tibial epiphyseal plates. The data were expressed as means SEM, * and ** stand for p < 0.05 and p, 0.01 between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group). (C) Chondrocytes layers of tibial epiphyseal plates. The data were expressed as means SEM, * and ** stand for p < 0.05 and p < 0.01 between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group). (D) A representative image of pathological changes in tibial epiphyseal plates (bar: 200 lm). a indicates columnar necrosis or lack, b indicates flake necrosis or lack and c indicates chondrocyte mass. (E) Pathological changes of tibial epiphyseal plates in four groups. The data were expressed as means SEM, * and *** stand for p < 0.05 and p < 0.001 between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group).
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significant lower value in both tibial and femur epiphyseal plates compared with SeS rats (Fig. 2B). In F1 generation, the proliferative layer of epiphyseal plate in SeD group was significant less than that in SeS group (Fig. 2C). In order to prove the Se deficient diet causing cartilage lesions in DA rats, pathology observation and statistics of epiphyseal plate injury were performed as a gold indicator. All femoral head and knee joint histologic sections were carried on H&E staining to observe cartilage lesions (Fig. S1). There were three specific representative lesions used for statistics: columnar necrosis or lack (Fig. 2D.a), flake necrosis or lack (Fig. 2D.b) and chondrocyte cluster (Fig. 2D.c). According to the staining and statistics results, there was no evidence of chondronecrosis in the superficial zone or the deep zone in the femoral head articular cartilage from the Se sufficient and Se deficient rats of both two generations. So we put the focus on the pathology observation of epiphyseal plates. Concerning the columnar necrosis or lack, SeD rats from both generations presented significant higher compared SeS ones (Fig. 2E). Analogously, SeD rats presented a remarkable higher level of flake necrosis or lack than SeS group (Fig. 2E). Se deficiency affects gene expression of extracellular matrix metabolism
COL II protein expression was determined via immunohistochemical (IHC). In both generations, SeD rats presented lower levels of COL II than SeS ones, significantly (Fig. 3A,B). F1 generation SeD group showed a lower COL II level than SeS group significantly, while no statistically significant difference in the F0 groups (Fig. 3C). In consideration of F1 generation possibly presented diverse relevant gene expression of cartilage compared to their parental generation under the Se deprivation stress, we tested more gene mRNA level in F1 rat’s cartilage. COL IIa1, COLXa1, ADAMTS-4, ADAMTS-5 were detected as cartilage development and metabolism representative genes (Fig. 3D–G). Only did ADAMTS-4 show a significantly up-regulation in SeD group (Fig. 3F), prompting there is more proteoglycan degradation. Se deficiency affects gene expression of GPx
Given these, we investigated GPx1 protein expression via visual IHC in the joint cartilage sections. As diagrams showed, in both generations, GPx1 expression in SeD rat tibia epiphyseal plates is significantly lower than that in the SeS ones (Fig. 4A,B). © 2015 APMIS. Published by John Wiley & Sons Ltd
In all four groups, the GPx1 and GPx4 mRNA expression levels in articular cartilage were detected (Fig. 4C,E). In F0 generation, GPx1 level of SeD rats showed a significant down regulation and faint correlation with the epiphyseal plate injury counting (Fig. 4C,D). Both SeD groups showed a significant down regulation of GPx4 than the SeS ones (Fig. 4E). These results revealed that Se deficiency can cause epiphyseal plate chondrocytes anabolism dissonance and show a lower oxidation resisting capacity. And epiphyseal plate lesion may occur for this.
DISCUSSION Se deficiency is deemed to be a major causative agent for KBD in humans (4, 12). In vivo Se status is commonly representative by plasma Se, plasma SePP1 concentration and plasma GPx activity, which suggest the Se intake from diet (18). Combining these two points, rats of two generations were fed with total synthesis of artificial Se deficient or sufficient diet so as to reserve Se level as only the experimental factor. Considering epidemiologic fact that KBD frequently affects adolescents and children (19, 20), we expressly used two generation DA rats. But, the metabolic balance of Se maybe relies on unsealed compensatory mechanism. It is well known that the most important function of Se is anti-oxidative stress, and KBD occurs as a consequence of oxidative damage to chondrocytes when associated with Se deficiency because of decreased antioxidant defence. The synergy of obstacle of Se-related genes expression and disordering metabolism of articular chondrocytes ECM were inducting apoptosis of chondrocyte, which contribute to cartilage destruction in KBD (21, 22). Like the SD rats experiments described by Fang Guan etc. (4), there was no evidence of chondronecrosis in the superficial zone or the deep zone in the femoral head articular cartilage from the Se sufficient and Se deficient DA rats of both two generations. So we put the focus on the pathology observation of epiphyseal plate. As both histological and biochemical investigated, the phenotypes are the same as the symptom of KBD patients, including metabolism disordering of articular cartilage, epiphyseal dysplasia, delayed skeletal development, secondary osteoarthritis (23). Comparing with SD rats treated with selenium deficient diet for 4 weeks (4), selenium deficient DA rats showed significant changes in the morphology of epiphyseal plate (Fig. 2). It seems that DA rats
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Fig. 3. The expression of collagens and ADAMTSs in cartilage. (A) Representative immunohistochemical images of tibial epiphyseal plates stained with antibody against type II collagen. (B) The comparison of type II collagen expression by immunohistochemistry. (C) mRNA expression of COLIIa1 by RT-qPCR. (D) mRNA expression of COLIa1 by RT-qPCR. (E) mRNA expression of COLXa1 by RT-qPCR. (F) mRNA expression of ADAMTS-4 by RT-qPCR. (G) mRNA expression of ADAMTS-5 by RT-qPCR. The data were expressed as means SEM, * and ** stand for p < 0.05 and p < 0.01 respectively between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group).
possess more higher sensitivity to low selenium. As for Wistar rats, necrosis of chondrocytes and dysfunctions of bone development were noticed 702
12 weeks after multifactorial exposure to the KBD endemic area diet, which was similar to the pathological changes seen in KBD (8). However, single © 2015 APMIS. Published by John Wiley & Sons Ltd
APMIS 123: 697–705
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12404
Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats ZIXIN MIN,1 WENXIANG ZHAO,1 NANNAN ZHONG,1 YUANXU GUO,1 MENGYAO SUN,1 QUANCHENG WANG,1 RUI ZHANG,1 JIDONG YAN,1 LIFANG TIAN,1 FUJUN ZHANG,1 YAN HAN,1 QILAN NING,1 LIESU MENG,1 JIAN SUN1,2 and SHEMIN LU1,2 1
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an; and 2Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China
Min Z, Zhao W, Zhong N, Guo Y, Sun M, Wang Q, Zhang R, Yan J, Tian L, Zhang F, Han Y, Ning Q, Meng L, Sun J, Lu S. Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats. APMIS 2015; 123: 697–705. This study aimed to observe the effects of Se deficiency on epiphyseal plates of two generation DA rats fed with artificial total synthetic low Se diet. All F0 and F1 DA rats were fed with synthetic low Se diet (SeD group) and low Se diet supplied with Se (SeS group). The levels of selenium and enzyme activities of GPx were detected in plasma of the rats. General growth of bone and articular cartilage was measured macroscopically and microscopically. The epiphyseal plate of femur heads or tibia were obtained to histological and immunohistochemical examinations. The cartilage from left knee joints and femur heads was used to detect the gene expression of collagens, ADAMTSs and several selenoproteins by RT-qPCR. Two generation SeD rats showed Se insufficiency status. The thicknesses of the femur and tibial epiphyseal plates in both F0 and F1 SeD rats were significantly less than that of SeS rats. In F1 generation, SeD rats showed much fewer proliferative chondrocyte layers than SeS ones. Importantly, two generation SeD rats both showed significantly more serious pathological changes of epiphyseal plates. In two generation rats, gene expressions of COL II, GPx1 and GPx4 were significantly down-regulated in SeD rats than SeS ones; meanwhile ADAMTS-4 showed an up-regulated expression in cartilage. Dietary Se deficiency can apparently cause epiphyseal plate lesion and decrease cartilage type II collagen production and GPx1 activity in two generation DA rats fed with the artificial total synthesis low Se diet. Key words: DA rats; selenium deficiency; diet; cartilage lesion. Jian Sun and Shemin Lu, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China. e-mails: [email protected]; [email protected] or [email protected]
Kashin-Beck disease (KBD), an endemic osteoarthropathy in Northern China, is characterized by pathological chondronecrosis of multiple areas of hyaline cartilage including articular cartilage and growth plates from different peripheral joints of the children from endemic areas (1). The disease manifests aberrant metabolism of articular cartilage leading to delayed skeletal development, secondary osteoarthritis and severe disability (2, 3). Concerning etiology of the disease, a combination of different environmental factors has been under considerations, including mycotoxicosis, iodine deficiency and selenium deficiency (4). Selenium (Se) is an essential trace element incorporated into selenoproteins that are associated with Received 20 January 2015. Accepted 17 April 2015
diverse physiological or pathological processes such as development and inflammation in mammals. Intriguingly, osteo-chondroprogenitor-specific deletion of the selenocysteinyl tRNA gene leads to the phenotypes similar to KBD, particularly showing chondronecrosis and abnormal skeletal development in the mice (5). The evidence has documented that Se as well as selenoproteins is important to the development of osteochondral system. Further on, Se supplementation has been used in the prophylatics and therapy of KBD despite of the ambiguous mechanism (6). Several animal models have been used to intuitively mimic the pathological manifestation of Se-deprived cartilage disorder (5). Existing models for KBD include outbred SD or Wistar rats fed with Se deficient or supplementary mycotoxin or 697
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allele is significantly associated with lower GPxs enzyme activity and with higher KBD risk, it is confirmed a potential genetic risk factor with the disease. In conclusion, Se plays a crucial role in epiphyseal plate growth, and Se deficiency lead to reduced selenoproteins biosynthesis which may decrease oxidative stress and imbalance the anabolism and catabolism of EMC in chondrocytes. Because of the feature of high sensitivity to arthritis, it was the first time that DA rats were applied for the establishment of low selenium model. The risk factor condition give rise to epiphyseal dysplasia in DA rats, similarly with KBD patients. So, the DA rats are perfect matching for KBD model with Se deficient nutritional status, and helpful for further study of the pathogenesis and prevention of KBDlike symptom.
CONFLICT OF INTEREST The authors have no conflicts of interest to declare.
This work was supported by grants from the National Natural Science Foundation of China (Project No. 81371986, 81301598, and 81201426), Project funded by China Postdoctoral Science Foundation (Project No. 2014T70928 and 2013M530427).
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osteoarthritis endemic to Northwestern China. Kashin-Beck Disease. Arthritis Rheum 2010;62:771– 80. 22. Wu SX, Wang WZ, Zhang F, Wu CY, Dennis BS, Qu CJ, et al. Expression profiles of genes involved in apoptosis and selenium metabolism in articular cartilage of patients with Kashin-Beck osteoarthritis. Gene 2014;535:124–30. 23. Cao J, Li S, Shi Z, Yue Y, Sun J, Chen J, et al. Articular cartilage metabolism in patients with KashinBeck Disease: an endemic osteoarthropathy in China. Osteoarthritis Cartilage 2008;16:680–8. 24. Yan J, Zheng Y, Min Z, Ning Q, Lu S. Selenium effect on selenoprotein transcriptome in chondrocytes. Biometals 2013;26:285–96.
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Fig. S1. Representative images of tibial epiphyseal plates stained with hematoxylin and eosin (H&E). (A) F0 generation SeS group (B) F0 generation SeD group (C) F1 generation SeS group (D) F1 generation SeD group. (bar: 200 lm). Table S1. RT-qPCR primers used for rat chondrocyte relative genes
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© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12404
Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats ZIXIN MIN,1 WENXIANG ZHAO,1 NANNAN ZHONG,1 YUANXU GUO,1 MENGYAO SUN,1 QUANCHENG WANG,1 RUI ZHANG,1 JIDONG YAN,1 LIFANG TIAN,1 FUJUN ZHANG,1 YAN HAN,1 QILAN NING,1 LIESU MENG,1 JIAN SUN1,2 and SHEMIN LU1,2 1
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an; and 2Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China
Min Z, Zhao W, Zhong N, Guo Y, Sun M, Wang Q, Zhang R, Yan J, Tian L, Zhang F, Han Y, Ning Q, Meng L, Sun J, Lu S. Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats. APMIS 2015; 123: 697–705. This study aimed to observe the effects of Se deficiency on epiphyseal plates of two generation DA rats fed with artificial total synthetic low Se diet. All F0 and F1 DA rats were fed with synthetic low Se diet (SeD group) and low Se diet supplied with Se (SeS group). The levels of selenium and enzyme activities of GPx were detected in plasma of the rats. General growth of bone and articular cartilage was measured macroscopically and microscopically. The epiphyseal plate of femur heads or tibia were obtained to histological and immunohistochemical examinations. The cartilage from left knee joints and femur heads was used to detect the gene expression of collagens, ADAMTSs and several selenoproteins by RT-qPCR. Two generation SeD rats showed Se insufficiency status. The thicknesses of the femur and tibial epiphyseal plates in both F0 and F1 SeD rats were significantly less than that of SeS rats. In F1 generation, SeD rats showed much fewer proliferative chondrocyte layers than SeS ones. Importantly, two generation SeD rats both showed significantly more serious pathological changes of epiphyseal plates. In two generation rats, gene expressions of COL II, GPx1 and GPx4 were significantly down-regulated in SeD rats than SeS ones; meanwhile ADAMTS-4 showed an up-regulated expression in cartilage. Dietary Se deficiency can apparently cause epiphyseal plate lesion and decrease cartilage type II collagen production and GPx1 activity in two generation DA rats fed with the artificial total synthesis low Se diet. Key words: DA rats; selenium deficiency; diet; cartilage lesion. Jian Sun and Shemin Lu, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China. e-mails: [email protected]; [email protected] or [email protected]
Kashin-Beck disease (KBD), an endemic osteoarthropathy in Northern China, is characterized by pathological chondronecrosis of multiple areas of hyaline cartilage including articular cartilage and growth plates from different peripheral joints of the children from endemic areas (1). The disease manifests aberrant metabolism of articular cartilage leading to delayed skeletal development, secondary osteoarthritis and severe disability (2, 3). Concerning etiology of the disease, a combination of different environmental factors has been under considerations, including mycotoxicosis, iodine deficiency and selenium deficiency (4). Selenium (Se) is an essential trace element incorporated into selenoproteins that are associated with Received 20 January 2015. Accepted 17 April 2015
diverse physiological or pathological processes such as development and inflammation in mammals. Intriguingly, osteo-chondroprogenitor-specific deletion of the selenocysteinyl tRNA gene leads to the phenotypes similar to KBD, particularly showing chondronecrosis and abnormal skeletal development in the mice (5). The evidence has documented that Se as well as selenoproteins is important to the development of osteochondral system. Further on, Se supplementation has been used in the prophylatics and therapy of KBD despite of the ambiguous mechanism (6). Several animal models have been used to intuitively mimic the pathological manifestation of Se-deprived cartilage disorder (5). Existing models for KBD include outbred SD or Wistar rats fed with Se deficient or supplementary mycotoxin or 697
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iodine (7, 8). However, the replication of the typical pathological phenotypes of KBD, such as deep chondrocyte necrosis and epiphyseal dysplasia, in the rats by only Se deficiency alone has been proven to be elusive (9). When Wistar rats were used, the same protocol seems to induce an putative osteopenia (10), as the same as in mice (11). Dark Agouti (DA) rats, an inbred strain, are more sensitive to various autoimmune joint diseases and commonly used for investigating the pathogenesis of rheumatoid arthritis (RA). DA rat models include pristane induced arthritis (PIA) (12), adjuvant-oil induced arthritis (13), CXI induced arthritis (CXIIA) and CII induced arthritis (CIIIA) (14, 15). Taking into consideration of much more sensitive to induced arthritis, DA rats were chosen to establish an animal model for KBD, hoping to observe more obvious effects of Se deficiency on cartilage pathology. In this study, we imitated the nutriture of the children from KBD endemic areas, and two generation DA rats were fed with Se deficient diet, to observe effect of Se deficiency on cartilage. We confirmed that Se deficient diet can impact the epiphyseal plate development and induce epiphyseal abnormality, and Se supplementation has a protective role in DA rats. The probable mechanism is related to regulating gene expression of antioxidant and extracellular matrix metabolism relevant molecules in chondrocytes. MATERIALS AND METHODS Rats Dark Agouti rats were bred and maintained in a specific pathogen-free animal house of our department. Age and sex-matched rats were used within all experiments, and each group contained 8 to 10 rats at age of 8 to 12 weeks. The experiments were approved by the Institutional Animal Ethics Committee of the university.
Diets Artificial total synthesis Se deficient and Se sufficient diets were made as R.F. Burk formula (16), and stored in fridge for use of only one week since multi-gelation should be avoided. Se contents were determined to ensure that Se intake from the diets was up to the experimental requirement. The Se sufficient diet was prepared by adding sodium selenite into the Se deficient one.
Animal experiments In F0 generation experiments, 20 DA rats at the age of 8 to 12 weeks were randomly divided into two groups matched by age and sex. Each group had six male and four female rats. The rats in Se sufficient (SeS) group were fed with Se sufficient diet as a control, and the rats in Se deficient (SeD) group were fed with Se deficient diet for 2 months.
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One male F0 rat and 2 to 3 female rats were kept in the same cage and fed with SeS or SeD diet. The produced offsprings would be used in F1 generation experiments. In F1 generation experiments, after weaning the rats were divided into 2 groups fed with SeS and SeD for 2 to 3 months, respectively. In the end of experiments, blood was collected from abdominal aorta to euthanize the rats. And hearts, livers, spleens and kidneys were collected and weighted immediately. All tissues were sampled to fix in the 4% paraformaldehyde for sectioning or to keep in 80 °C for RNA isolation. After removing muscle and other soft tissues, left low limbs were quickly placed in the fixing buffer, while the right femur heads were saved in 80 °C for RNA isolation.
Detection of Se content and GPx activity Contents of Se in diets and sera of the DA rats were determined by fluorometric method fluorescent atomic absorption spectrometry. Total plasma glutathione peroxidase (GPx) activity was measured by using the direct DTNB method.
Histology staining Removed livers, hearts, spleens and kidneys from each rat were fixed in 4% paraformaldehyde in PBS solution and embedded together in paraffin. Knee joints fixed with 4% paraformaldehyde (17) were tenderly decalcified in 10% EDTA liquid for 4 week, subsequently paraffin-embedded for staining to observe morphological changes of epiphyseal plate. Then all sections were cut at 5 lm thickness and stained with hematoxylin and eosin (H&E), otherwise, safranin O and fast green.
Measurement of growth plate and chondrocytes The thickness and chondrocyte layers of epiphyseal plate were measured under light microscope. The images of each epiphyseal plate on sections were obtained under the microscope (Olympus BX51; Olympus, Tokyo, Japan) equipped with a digital camera (Olympus PD71; Olympus). All images should cover the whole structure of epiphyseal plates after merging. Five sites evenly locating in each epiphyseal plate were selected to measure thickness and chondrocyte layers and the average value was obtained to stand for each animal. These values were all calculated by Image-Pro Plus 6 (Media Cybernetics, Silver Spring, MD, USA). Also, femur diameters, femur lengths and tibial lengths were measured by vernier caliper.
Assessment of epiphyseal plate lesion Morphological changes of epiphyseal plate were observed and the abnormal lesions can be divided into three types: column necrosis or missing is defined as a necrosis or only chondrocyte lacuna in a whole column chondrocytes from proliferation layer to the hypertrophy layer; laminar necrosis or missing is defined as necrosis or chondrocyte lacuna in one or a few layers of continuous several columns (at least 2); and chondrocyte cluster is defined as round or elliptical or irregular round cell cluster without adjacent chondrocyte column and lacking at least 2 layer of cells between the upper and the nether layer. © 2015 APMIS. Published by John Wiley & Sons Ltd
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Total RNA extraction Total RNA was isolated from femur head cartilage or internal organ samples using the TRIzolÒ method. Two microgram of each total RNA from cartilage was timely reversed to cDNA according to the manufacturer’s instruction (RevertAidTM; Fermentas Life Sciences, Burlington, ON, Canada) and stored at 20 °C until used.
Real-time quantitative PCR analysis
gene expression assay analysis were calculated by using Student’s T test. Correlation analysis was performed by Spearman test. In all tests a value of p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001). All analyses were performed using the software program GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
RESULTS
The mRNA expressions were tested by Real-time quantitative PCR (RT-qPCR), which was performed on iQ5 realtime PCR detection system (Bio-Rad, Hercules, CA, USA) with SYBRÒ Premix Ex TaqTM II (TaKaRa, Shiga, Japan). The relative gene expression is normalized by GAPDH. The information of primers is depicted in Table S1.
Immunohistochemistry staining Knee joints fixed with 4% paraformaldehyde (17) were decalcified in 10% EDTA liquid for 4 week, subsequently paraffin-embedded and dissected at 5 lm thickness. After intrinsic peroxidase activity was blocked with 3% hydrogen peroxide, the sections were incubated with 1.5% BSA for 1 h. Then sections were covered with 1:200 dilution of polyclonal antibody against type II collagen a1 (COL II a1) and 1:250 monoclonal antibody against GPx1 respectively, for 14 h at 4 °C wet box in fridge. The sections were rinsed and incubated sequentially with biotinylated secondary antibody for 1 h and ABC reagent (Vector Laboratories, Burlingame, CA, USA) for 10 min at room temperature. Rabbit IgG was used as a negative control.
Statistical analysis Data were represented as means SEM. The pathological data were calculated using Mann–Whitney U test, and
Artificial total synthetic Se deficient diet can cause Se insufficiency status of rats
As expected, the Se content in Se deficient synthetic diet was significantly lower than that in Se supplemental one (Table 1). This result confirmed that the diet with Se deficiency can be used for subsequent experiments. The plasma Se contents showed much lower in SeD rats than SeS ones (Table 1). Plasma total GPx activity of the SeD rats showed a significant lower level than the SeS group rats in both F0 and F1 generations, respectively (Fig. 1A). The expressions of liver GPx1 and SEPP1 in the SeD group were both significantly lower than the SeS one (Fig. 1B,C). All these indicated that the rats fed with SeD diet existed in a Se deficient status. Se deficiency can cause epiphyseal plate lesion
As an index of cartilage development, the thickness of tibial and femur epiphyseal plates were overall measured after safranin O and fast green staining (Fig. 2A). In F0 generation, the SeD rats showed a
Table 1. Selenium contents in rat diet and blood samples of each group [mean SEM (n)] Groups Se content in diet (lg/g) Se content in blood (lg/mL) F0 rats F1 rats Se sufficient 0.288 0.038 (10) 0.676 0.044 (10) 0.520 0.054 (8) Se deficient 0.018 0.005 (10)*** 0.262 0.026 (10)*** 0.032 0.009 (6)*** ***p < 0.001 when compared two groups. Se levels were measured by fluorescent atomic absorption spectrometry.
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Fig. 1. Evaluation of Se status in experimental rats. (A) Plasma total GPx activity in each group. The data were expressed as means SEM, * and ** stand for p < 0.05 and 0.01 respectively between two groups (F0 group: n = 10 for each group; F1 group: n = 8 for SeS group, and n = 6 for SeD group). (B) and (C) mRNA expression of GPx1 and SEPP1 in rat livers from F1 group. The data were expressed as means SEM, * stands for p < 0.05 between two groups (F1 rats: n = 8 for SeS group, and n = 6 for SeD group). © 2015 APMIS. Published by John Wiley & Sons Ltd
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Fig. 2. Comparisons of bone and cartilage growth in F0 and F1 rats among 4 groups. (A) Representative images of tibial epiphyseal plates from 4 experimental groups. The sections of rat knee joints were stained with Safranine O and fast-green dye (bar: 200 lm). (B)The thicknesses of femur and tibial epiphyseal plates. The data were expressed as means SEM, * and ** stand for p < 0.05 and p, 0.01 between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group). (C) Chondrocytes layers of tibial epiphyseal plates. The data were expressed as means SEM, * and ** stand for p < 0.05 and p < 0.01 between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group). (D) A representative image of pathological changes in tibial epiphyseal plates (bar: 200 lm). a indicates columnar necrosis or lack, b indicates flake necrosis or lack and c indicates chondrocyte mass. (E) Pathological changes of tibial epiphyseal plates in four groups. The data were expressed as means SEM, * and *** stand for p < 0.05 and p < 0.001 between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group).
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significant lower value in both tibial and femur epiphyseal plates compared with SeS rats (Fig. 2B). In F1 generation, the proliferative layer of epiphyseal plate in SeD group was significant less than that in SeS group (Fig. 2C). In order to prove the Se deficient diet causing cartilage lesions in DA rats, pathology observation and statistics of epiphyseal plate injury were performed as a gold indicator. All femoral head and knee joint histologic sections were carried on H&E staining to observe cartilage lesions (Fig. S1). There were three specific representative lesions used for statistics: columnar necrosis or lack (Fig. 2D.a), flake necrosis or lack (Fig. 2D.b) and chondrocyte cluster (Fig. 2D.c). According to the staining and statistics results, there was no evidence of chondronecrosis in the superficial zone or the deep zone in the femoral head articular cartilage from the Se sufficient and Se deficient rats of both two generations. So we put the focus on the pathology observation of epiphyseal plates. Concerning the columnar necrosis or lack, SeD rats from both generations presented significant higher compared SeS ones (Fig. 2E). Analogously, SeD rats presented a remarkable higher level of flake necrosis or lack than SeS group (Fig. 2E). Se deficiency affects gene expression of extracellular matrix metabolism
COL II protein expression was determined via immunohistochemical (IHC). In both generations, SeD rats presented lower levels of COL II than SeS ones, significantly (Fig. 3A,B). F1 generation SeD group showed a lower COL II level than SeS group significantly, while no statistically significant difference in the F0 groups (Fig. 3C). In consideration of F1 generation possibly presented diverse relevant gene expression of cartilage compared to their parental generation under the Se deprivation stress, we tested more gene mRNA level in F1 rat’s cartilage. COL IIa1, COLXa1, ADAMTS-4, ADAMTS-5 were detected as cartilage development and metabolism representative genes (Fig. 3D–G). Only did ADAMTS-4 show a significantly up-regulation in SeD group (Fig. 3F), prompting there is more proteoglycan degradation. Se deficiency affects gene expression of GPx
Given these, we investigated GPx1 protein expression via visual IHC in the joint cartilage sections. As diagrams showed, in both generations, GPx1 expression in SeD rat tibia epiphyseal plates is significantly lower than that in the SeS ones (Fig. 4A,B). © 2015 APMIS. Published by John Wiley & Sons Ltd
In all four groups, the GPx1 and GPx4 mRNA expression levels in articular cartilage were detected (Fig. 4C,E). In F0 generation, GPx1 level of SeD rats showed a significant down regulation and faint correlation with the epiphyseal plate injury counting (Fig. 4C,D). Both SeD groups showed a significant down regulation of GPx4 than the SeS ones (Fig. 4E). These results revealed that Se deficiency can cause epiphyseal plate chondrocytes anabolism dissonance and show a lower oxidation resisting capacity. And epiphyseal plate lesion may occur for this.
DISCUSSION Se deficiency is deemed to be a major causative agent for KBD in humans (4, 12). In vivo Se status is commonly representative by plasma Se, plasma SePP1 concentration and plasma GPx activity, which suggest the Se intake from diet (18). Combining these two points, rats of two generations were fed with total synthesis of artificial Se deficient or sufficient diet so as to reserve Se level as only the experimental factor. Considering epidemiologic fact that KBD frequently affects adolescents and children (19, 20), we expressly used two generation DA rats. But, the metabolic balance of Se maybe relies on unsealed compensatory mechanism. It is well known that the most important function of Se is anti-oxidative stress, and KBD occurs as a consequence of oxidative damage to chondrocytes when associated with Se deficiency because of decreased antioxidant defence. The synergy of obstacle of Se-related genes expression and disordering metabolism of articular chondrocytes ECM were inducting apoptosis of chondrocyte, which contribute to cartilage destruction in KBD (21, 22). Like the SD rats experiments described by Fang Guan etc. (4), there was no evidence of chondronecrosis in the superficial zone or the deep zone in the femoral head articular cartilage from the Se sufficient and Se deficient DA rats of both two generations. So we put the focus on the pathology observation of epiphyseal plate. As both histological and biochemical investigated, the phenotypes are the same as the symptom of KBD patients, including metabolism disordering of articular cartilage, epiphyseal dysplasia, delayed skeletal development, secondary osteoarthritis (23). Comparing with SD rats treated with selenium deficient diet for 4 weeks (4), selenium deficient DA rats showed significant changes in the morphology of epiphyseal plate (Fig. 2). It seems that DA rats
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Fig. 3. The expression of collagens and ADAMTSs in cartilage. (A) Representative immunohistochemical images of tibial epiphyseal plates stained with antibody against type II collagen. (B) The comparison of type II collagen expression by immunohistochemistry. (C) mRNA expression of COLIIa1 by RT-qPCR. (D) mRNA expression of COLIa1 by RT-qPCR. (E) mRNA expression of COLXa1 by RT-qPCR. (F) mRNA expression of ADAMTS-4 by RT-qPCR. (G) mRNA expression of ADAMTS-5 by RT-qPCR. The data were expressed as means SEM, * and ** stand for p < 0.05 and p < 0.01 respectively between two groups (F0 rats: n = 10 for each group; F1 rats: n = 8 for SeS group, and n = 6 for SeD group).
possess more higher sensitivity to low selenium. As for Wistar rats, necrosis of chondrocytes and dysfunctions of bone development were noticed 702
12 weeks after multifactorial exposure to the KBD endemic area diet, which was similar to the pathological changes seen in KBD (8). However, single © 2015 APMIS. Published by John Wiley & Sons Ltd
APMIS 123: 697–705
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12404
Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats ZIXIN MIN,1 WENXIANG ZHAO,1 NANNAN ZHONG,1 YUANXU GUO,1 MENGYAO SUN,1 QUANCHENG WANG,1 RUI ZHANG,1 JIDONG YAN,1 LIFANG TIAN,1 FUJUN ZHANG,1 YAN HAN,1 QILAN NING,1 LIESU MENG,1 JIAN SUN1,2 and SHEMIN LU1,2 1
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an; and 2Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China
Min Z, Zhao W, Zhong N, Guo Y, Sun M, Wang Q, Zhang R, Yan J, Tian L, Zhang F, Han Y, Ning Q, Meng L, Sun J, Lu S. Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats. APMIS 2015; 123: 697–705. This study aimed to observe the effects of Se deficiency on epiphyseal plates of two generation DA rats fed with artificial total synthetic low Se diet. All F0 and F1 DA rats were fed with synthetic low Se diet (SeD group) and low Se diet supplied with Se (SeS group). The levels of selenium and enzyme activities of GPx were detected in plasma of the rats. General growth of bone and articular cartilage was measured macroscopically and microscopically. The epiphyseal plate of femur heads or tibia were obtained to histological and immunohistochemical examinations. The cartilage from left knee joints and femur heads was used to detect the gene expression of collagens, ADAMTSs and several selenoproteins by RT-qPCR. Two generation SeD rats showed Se insufficiency status. The thicknesses of the femur and tibial epiphyseal plates in both F0 and F1 SeD rats were significantly less than that of SeS rats. In F1 generation, SeD rats showed much fewer proliferative chondrocyte layers than SeS ones. Importantly, two generation SeD rats both showed significantly more serious pathological changes of epiphyseal plates. In two generation rats, gene expressions of COL II, GPx1 and GPx4 were significantly down-regulated in SeD rats than SeS ones; meanwhile ADAMTS-4 showed an up-regulated expression in cartilage. Dietary Se deficiency can apparently cause epiphyseal plate lesion and decrease cartilage type II collagen production and GPx1 activity in two generation DA rats fed with the artificial total synthesis low Se diet. Key words: DA rats; selenium deficiency; diet; cartilage lesion. Jian Sun and Shemin Lu, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China. e-mails: [email protected]; [email protected] or [email protected]
Kashin-Beck disease (KBD), an endemic osteoarthropathy in Northern China, is characterized by pathological chondronecrosis of multiple areas of hyaline cartilage including articular cartilage and growth plates from different peripheral joints of the children from endemic areas (1). The disease manifests aberrant metabolism of articular cartilage leading to delayed skeletal development, secondary osteoarthritis and severe disability (2, 3). Concerning etiology of the disease, a combination of different environmental factors has been under considerations, including mycotoxicosis, iodine deficiency and selenium deficiency (4). Selenium (Se) is an essential trace element incorporated into selenoproteins that are associated with Received 20 January 2015. Accepted 17 April 2015
diverse physiological or pathological processes such as development and inflammation in mammals. Intriguingly, osteo-chondroprogenitor-specific deletion of the selenocysteinyl tRNA gene leads to the phenotypes similar to KBD, particularly showing chondronecrosis and abnormal skeletal development in the mice (5). The evidence has documented that Se as well as selenoproteins is important to the development of osteochondral system. Further on, Se supplementation has been used in the prophylatics and therapy of KBD despite of the ambiguous mechanism (6). Several animal models have been used to intuitively mimic the pathological manifestation of Se-deprived cartilage disorder (5). Existing models for KBD include outbred SD or Wistar rats fed with Se deficient or supplementary mycotoxin or 697
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allele is significantly associated with lower GPxs enzyme activity and with higher KBD risk, it is confirmed a potential genetic risk factor with the disease. In conclusion, Se plays a crucial role in epiphyseal plate growth, and Se deficiency lead to reduced selenoproteins biosynthesis which may decrease oxidative stress and imbalance the anabolism and catabolism of EMC in chondrocytes. Because of the feature of high sensitivity to arthritis, it was the first time that DA rats were applied for the establishment of low selenium model. The risk factor condition give rise to epiphyseal dysplasia in DA rats, similarly with KBD patients. So, the DA rats are perfect matching for KBD model with Se deficient nutritional status, and helpful for further study of the pathogenesis and prevention of KBDlike symptom.
CONFLICT OF INTEREST The authors have no conflicts of interest to declare.
This work was supported by grants from the National Natural Science Foundation of China (Project No. 81371986, 81301598, and 81201426), Project funded by China Postdoctoral Science Foundation (Project No. 2014T70928 and 2013M530427).
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Fig. S1. Representative images of tibial epiphyseal plates stained with hematoxylin and eosin (H&E). (A) F0 generation SeS group (B) F0 generation SeD group (C) F1 generation SeS group (D) F1 generation SeD group. (bar: 200 lm). Table S1. RT-qPCR primers used for rat chondrocyte relative genes
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