$ournal o~ Tongfi Medical university 10(2)'100-I03, 1990
100
Cordyeeps Sinensis-I as an Immunosuppressant i n Heterotopic Heart Allograft Model in Rats
ZHANG Zheng (gg ~J~), XIA Sui-shen (j~r
Institute ol Organ Transplantation, Tongji Hospital, Tongfi Medical Uni~arsity, Wuhan
Summary.. To investigate the inhibitory effects of Cordyeeps Sinensis--I (CS-1) on the immune response responsible for the organ transplant rejection, we studied its effects of prolonging heterotopie heart allograft survival on rat model with heart graft; the effects of CS-I were compared with those of eyelosporine A and glueoeortieoid, and the possible pathological changes caused by CS-I were observed. Our results showed that CS-1 can prolong the survival of grafted heart without causing infection, and it did not exex~t detrimental effects on vital organs. As a result, it proves to be a promising immunosuppressant in clinical organ transplantation in the future. Key words,
Cordyeeps Sinensis, heterotopic heart transplantation, suppressant
Cordyceps S i n e n s i s - I ( C S - 1 ) is a fermented product of the f u n g u s P a e c i lomyces H e p i a l i Chen, w h i c h w a s s e parated f r o m natural Cordyceps S i n e n s i s , a w e l l - k n o w n v a l u a b l e Chinese t r a d i t i o n a l remedy m a i n l y collected f r o m Qinghai Province, China. Recently it w a s s h o w n that CS-1 can i n h i b i t cellular i m m u n e response a n d prolong the s u r v i v a l of the allografted cardiac muscle t i s s u e s in mice. I n s p i r e d b y these f i n d i n g s , we i n v e s t i g a t e d the a n t i rejection effects of CS-1 in organ t r a n s p l a n t a t i o n in small a n i m a l s . I n t h i s experiment we u s e d heterotopic heart allograft as model to evaluate the e f fects of CS-1 on mean s u r v i v a l time
immuno-
( M S T ) and to compare the effects of CS-1 w i t h those of c y c l o s p o r i n e A ( C s A ) and glucocorticoid ( G C ) . Effects of CS-1 on some v i t a l organs were also observed. MATERIALS AND METHODS Animals Inbred rats w e i g h i n g 200-300 g p r o v i d e d b y the A n i m a l C e n ter of T o n g j i Medical U n i v e r s i t y were used w i t h W i s t a r as donors and SD as recipients. Heterotopie heart transplantation I n t r a a b d o m i n a l a u x i l i a r y heart t r a n s p l a n t a t i o n w a s performed according to the method described b y One iii and Lee zj w i t h slight m o d i f i c a t i o n s . B r i e f -
Journal of Tongji Medical University 10(2):100-103, 1990 101
ly, after ether anesthesia the donor aorta and pulmonary artery were anastomosed end-to-side to the abdominal aorta and vena cava of the recipient respectively. Ischemic time of heart graft was about 45 r a i n The day of operation was taken as day 0, the day of cessation of palpable heart contraction was taken as the endpoint of allograft survival. Posttransplantive observation The heart transplants were examined by daily abdominal palpation and regular ECG monitoring. Determination of cndpoint of allograft survival was based on 1) palpation revealing no heartbeating; 2) ECG showing loss of electric a c t i v i t y of the grafted heart~ 3) exploratory laparotomy showing no contraction of cardiac muscle; and 4) h i s topathological evaluation. The drugs used in immunosuppressive therapy were as follows: 1) CS-1 (Jiangxi Chinese Medicine Factory) was dissolved in 0 . 9 ~ sodium chloride to a concentration of 4 ~ and 16~b. 2) CsA (Sandoz) was dissolved in 0.9 0~ sodium chloride to a concentration of 1 mg/ml. 3) GC for injection was dissolved in 0.90~ sodium chloride to a concentration of 5 me/m1. The drugs mentioned above were administered by i.p. injection in d i f ferent doses (table 1) on day -4, -2, 0,2,4,6. Experimental design and statistical analysis 1. The rats were divided into 5 Table 2.
Table 1.
Experimental design
Group (No. of rats)
Dose
N S (12) CsA (12)
3ml 3ml (10mg/kg)
GC (12) 16~ CS-1 (11) 4o~ CS-I (12)
3ml (30mg/kg) 3ml (1.6g/kg) 3ml (0.4g/kg)
groups according to the drugs used, including control group receiving norreal saline (NS; table 1) to observe the effects of CS-1 on MS]? of heterotopic heart graft. The MST was expressed as +sx. The statistical significance of the results in the experimental groups was calculated by t test. 2. The effects of CS-1 on liver, kidney, spleen and thymus of rats were also histopathologically investigated. 6 rats were given 4 ~ CS-1 i.p. at 0.4 g/kg once per day for 5 consecutive days, without receiving transplantation. Thereafter the kidneys, liver, spleen and thymus were removed for histopathological study. 18 rats given NS, CsA or GC (6 rats respectively) were used as controls. RESULTS
In our experiment, 154 hcterotopic heart transplantations were performed including 96 preparatory and 60 formal experiments. In the 60 formal experiments, 17 rats died unexpectedly duc to various causes such as operation injury, postoperative peritoneal infection, ctc (table 2).
Causes of unexpected death
(NoGroup of rats)
Anesthesia
Operation injury
Infection
NS(12)
I
I
2
CsA(I 2)
1
2
i 4
GC(12 16~ CS-I(I I)
I
4~ CS-1(13)
1
1
Other causes
102
o
u~ o
V
A
o
t~ 0
o
L~ 0
0
o
0
0
v
A
v
V
o 0
§
0
e-
e~ o
o
+1
+1
+1
,.-i
III
o
A A
g A
~g
A
A r
"r
The results were summarized as follows: As s h o w n in table 3, in the groups given 40~ and 160~ CS-1, MST of the grafted heart was prolonged s i g n i ficantly ( P ~ 0 . 0 1 and P ~ 0 . 0 5 respect i v e l y ) , as compared w i t h the group g i ven NS. In none of the CS-1 groups occurred postoperative peritoneal infection. There were four cases w i t h survival time of more than 90 days. The h i s t o pathological observation revealed differ ~ ences in histologic appearance between CS-1 groups and NS group. The CS-1 groups showed chronic rejection, while the NS group demonstrated serious acute rejection. MST of 4 ~ CS-1 group was o b v i o u s l y longer than that of CsA group, though the difference between them was not statistically significant ( P ~ 0 . 0 5 ) . Compared w i t h GC group, MST of the grafted heart of 4 ~ CS-1 group was prolonged significantly ( P ~ 0 . 0 5 ) ; no infection was observed. In 4 animals of the GC group complicating peritoneal infection occurred. There was no significant difference in MST between the 1 6 ~ CS-1 group and GC group. The histopathological study of rats receiving CS-1 or NS w i t h o u t undergoing transplantation showed that CS-1 did not exert apparent detrimental effects on liver, kidneys, spleen and thymus. DISCUSSION
0
~
r
"-" !
b,,
Cordyceps Sinensis is growing abundantly in the provinces Qinghai, Yunnan, Sichuan and Hunan in a v a r iety of species. In the I950s and 1960s, its chemical components and pharmaceutical effects were reported in the literature at home and abroad, s h o w i n g that it possessed bronchodilating, sedative and sleep-inducing, bacteriostatic and anti-cancerous effects but extremely low toxicity sj. Recently, there have been reports on its way of activating m o n o nuclear phagocyte system and enhancing the engulfing function of macropha-
Journal o! Tongji Medical University 10(2):100-103, 1990 103 ges TM.Huang et aY ~1 reported that c u l t u re of hyphae of Cordyceps Sinensis can obviously i nhi bi t E-rosette forming in humans and prolong the survival of skin a11ografts in mice and cardiac tissue transplantation into the mouse ear, suggesting that it is a very promising drug for anti-rejection therapy. So far there were no r e ports on its anti-rejection effects on animal organ transplantation. T hi s paper furnished evidence of its value in that respect. Our results demonstrated that CS-1 of a given concentration possesses d i s tinct anti-rejection effects. Considering the mechanism of rejection in organ transplantation, we postulate that the anti-rejection effects of CS-1 might be related to i t s i n h i b i t o r y effects on helper T cells and enhancing effects on suppressor T cells. Wang et al ~a~ reported that CS-1 could i n h i b i t T lymphocyte blastogenesis of spleen lymphocytes from different strains of mice b y ConA and PHA, markedly enhance the function of suppressor T cells induced by ConA in vi t r o and apparently enhance the function of spontaneous suppressor T lymphocytes in v i v o . These findings support our postula-
tion. Our experimental results also s u g gested that CS-1 exerted no detrimental effects o n spleen and thymus of rats, and it did not demonstrate h e p a t o t o x i city and nephrotoxicity. In addition, it could effectively prevent rejection and prolong the s u r v i v a l of grafts without causing infection. All these findings suggested that the anti-rejection effects of CS-1 are selective. The good anti-rejection effects of CS-1 might be related to its induction of immuno-tolerance. According to l i terature t~ the allografts of rats or mice were believed to develop immuno-tolerance if the s u r v i v a l time exceeded 60 days. In our experiment, 4 rats (30o~) survived over 90 days in 4o~ CS-1 group. We hold that the 4 rats
did develop immuno-tolerance to heart allografts. It is currently believed that suppressor T cells play an i m p o r tant role in the formation and maintenance of immunological tolerance. After their activation, they can produce a soluble i nhi bi t or of immune reaction, thereby i nhi bi t i ng function of the helper T cells and immune response of the body. CS-1 might induce i m m u n o tolerance by enhancing the function of the suppressor T cells. The anti-rejection effects of CS-1 apparently depended on the drug concentration: the effects of ~ CS-1 were obvi ousl y greater than those of 1 6 ~ CS-1. T hi s may be attributed to the dual effects of the immunosuppressant, the mechanism of which still awaits further investigation. From our experimental results, we are led to conclude that CS-1 is a new very promising immunosuppressant or immuno-regulator which might be a p plied in clinical allotransplantation in the future.
REFERENCES 1. One K, et al. Improved technique of heart transplantation in rat. J Thorac Cardiovasc Surg 1969;57:225"7. 2. Lee S, et al. A simplified model for hoterotopic rat heart transplantation. Transplanlation. 19~2~33:438-40. 3. T ~ , ~ , & ~ ~ l ~ . 4. ~,~,
~.
~:~z_.~_~r
,~,,~ 1984;9(4):248-50. S . ~,"~"~, ~ . ~ t ~ r IV. ~ ~ ~,~.~- ~ ~ ~ ~ , ] ~ ~r~ ~.. ~]~~ ' ~. ~ ~ ~[~ 19g@;17(5): 329-31. 6. ~&, ~. ~ ~ ~ 2 . v. 1988:17 (5) : 332-4. 9 Mayumi H, et al. Drug-induced tolerance to allografts in mice.- V. Prolongation of skirt graft survival in tolerant mice with combined immunosuppressive treatments. Transplantation 1985)39,$35-8.
100
Cordyeeps Sinensis-I as an Immunosuppressant i n Heterotopic Heart Allograft Model in Rats
ZHANG Zheng (gg ~J~), XIA Sui-shen (j~r
Institute ol Organ Transplantation, Tongji Hospital, Tongfi Medical Uni~arsity, Wuhan
Summary.. To investigate the inhibitory effects of Cordyeeps Sinensis--I (CS-1) on the immune response responsible for the organ transplant rejection, we studied its effects of prolonging heterotopie heart allograft survival on rat model with heart graft; the effects of CS-I were compared with those of eyelosporine A and glueoeortieoid, and the possible pathological changes caused by CS-I were observed. Our results showed that CS-1 can prolong the survival of grafted heart without causing infection, and it did not exex~t detrimental effects on vital organs. As a result, it proves to be a promising immunosuppressant in clinical organ transplantation in the future. Key words,
Cordyeeps Sinensis, heterotopic heart transplantation, suppressant
Cordyceps S i n e n s i s - I ( C S - 1 ) is a fermented product of the f u n g u s P a e c i lomyces H e p i a l i Chen, w h i c h w a s s e parated f r o m natural Cordyceps S i n e n s i s , a w e l l - k n o w n v a l u a b l e Chinese t r a d i t i o n a l remedy m a i n l y collected f r o m Qinghai Province, China. Recently it w a s s h o w n that CS-1 can i n h i b i t cellular i m m u n e response a n d prolong the s u r v i v a l of the allografted cardiac muscle t i s s u e s in mice. I n s p i r e d b y these f i n d i n g s , we i n v e s t i g a t e d the a n t i rejection effects of CS-1 in organ t r a n s p l a n t a t i o n in small a n i m a l s . I n t h i s experiment we u s e d heterotopic heart allograft as model to evaluate the e f fects of CS-1 on mean s u r v i v a l time
immuno-
( M S T ) and to compare the effects of CS-1 w i t h those of c y c l o s p o r i n e A ( C s A ) and glucocorticoid ( G C ) . Effects of CS-1 on some v i t a l organs were also observed. MATERIALS AND METHODS Animals Inbred rats w e i g h i n g 200-300 g p r o v i d e d b y the A n i m a l C e n ter of T o n g j i Medical U n i v e r s i t y were used w i t h W i s t a r as donors and SD as recipients. Heterotopie heart transplantation I n t r a a b d o m i n a l a u x i l i a r y heart t r a n s p l a n t a t i o n w a s performed according to the method described b y One iii and Lee zj w i t h slight m o d i f i c a t i o n s . B r i e f -
Journal of Tongji Medical University 10(2):100-103, 1990 101
ly, after ether anesthesia the donor aorta and pulmonary artery were anastomosed end-to-side to the abdominal aorta and vena cava of the recipient respectively. Ischemic time of heart graft was about 45 r a i n The day of operation was taken as day 0, the day of cessation of palpable heart contraction was taken as the endpoint of allograft survival. Posttransplantive observation The heart transplants were examined by daily abdominal palpation and regular ECG monitoring. Determination of cndpoint of allograft survival was based on 1) palpation revealing no heartbeating; 2) ECG showing loss of electric a c t i v i t y of the grafted heart~ 3) exploratory laparotomy showing no contraction of cardiac muscle; and 4) h i s topathological evaluation. The drugs used in immunosuppressive therapy were as follows: 1) CS-1 (Jiangxi Chinese Medicine Factory) was dissolved in 0 . 9 ~ sodium chloride to a concentration of 4 ~ and 16~b. 2) CsA (Sandoz) was dissolved in 0.9 0~ sodium chloride to a concentration of 1 mg/ml. 3) GC for injection was dissolved in 0.90~ sodium chloride to a concentration of 5 me/m1. The drugs mentioned above were administered by i.p. injection in d i f ferent doses (table 1) on day -4, -2, 0,2,4,6. Experimental design and statistical analysis 1. The rats were divided into 5 Table 2.
Table 1.
Experimental design
Group (No. of rats)
Dose
N S (12) CsA (12)
3ml 3ml (10mg/kg)
GC (12) 16~ CS-1 (11) 4o~ CS-I (12)
3ml (30mg/kg) 3ml (1.6g/kg) 3ml (0.4g/kg)
groups according to the drugs used, including control group receiving norreal saline (NS; table 1) to observe the effects of CS-1 on MS]? of heterotopic heart graft. The MST was expressed as +sx. The statistical significance of the results in the experimental groups was calculated by t test. 2. The effects of CS-1 on liver, kidney, spleen and thymus of rats were also histopathologically investigated. 6 rats were given 4 ~ CS-1 i.p. at 0.4 g/kg once per day for 5 consecutive days, without receiving transplantation. Thereafter the kidneys, liver, spleen and thymus were removed for histopathological study. 18 rats given NS, CsA or GC (6 rats respectively) were used as controls. RESULTS
In our experiment, 154 hcterotopic heart transplantations were performed including 96 preparatory and 60 formal experiments. In the 60 formal experiments, 17 rats died unexpectedly duc to various causes such as operation injury, postoperative peritoneal infection, ctc (table 2).
Causes of unexpected death
(NoGroup of rats)
Anesthesia
Operation injury
Infection
NS(12)
I
I
2
CsA(I 2)
1
2
i 4
GC(12 16~ CS-I(I I)
I
4~ CS-1(13)
1
1
Other causes
102
o
u~ o
V
A
o
t~ 0
o
L~ 0
0
o
0
0
v
A
v
V
o 0
§
0
e-
e~ o
o
+1
+1
+1
,.-i
III
o
A A
g A
~g
A
A r
"r
The results were summarized as follows: As s h o w n in table 3, in the groups given 40~ and 160~ CS-1, MST of the grafted heart was prolonged s i g n i ficantly ( P ~ 0 . 0 1 and P ~ 0 . 0 5 respect i v e l y ) , as compared w i t h the group g i ven NS. In none of the CS-1 groups occurred postoperative peritoneal infection. There were four cases w i t h survival time of more than 90 days. The h i s t o pathological observation revealed differ ~ ences in histologic appearance between CS-1 groups and NS group. The CS-1 groups showed chronic rejection, while the NS group demonstrated serious acute rejection. MST of 4 ~ CS-1 group was o b v i o u s l y longer than that of CsA group, though the difference between them was not statistically significant ( P ~ 0 . 0 5 ) . Compared w i t h GC group, MST of the grafted heart of 4 ~ CS-1 group was prolonged significantly ( P ~ 0 . 0 5 ) ; no infection was observed. In 4 animals of the GC group complicating peritoneal infection occurred. There was no significant difference in MST between the 1 6 ~ CS-1 group and GC group. The histopathological study of rats receiving CS-1 or NS w i t h o u t undergoing transplantation showed that CS-1 did not exert apparent detrimental effects on liver, kidneys, spleen and thymus. DISCUSSION
0
~
r
"-" !
b,,
Cordyceps Sinensis is growing abundantly in the provinces Qinghai, Yunnan, Sichuan and Hunan in a v a r iety of species. In the I950s and 1960s, its chemical components and pharmaceutical effects were reported in the literature at home and abroad, s h o w i n g that it possessed bronchodilating, sedative and sleep-inducing, bacteriostatic and anti-cancerous effects but extremely low toxicity sj. Recently, there have been reports on its way of activating m o n o nuclear phagocyte system and enhancing the engulfing function of macropha-
Journal o! Tongji Medical University 10(2):100-103, 1990 103 ges TM.Huang et aY ~1 reported that c u l t u re of hyphae of Cordyceps Sinensis can obviously i nhi bi t E-rosette forming in humans and prolong the survival of skin a11ografts in mice and cardiac tissue transplantation into the mouse ear, suggesting that it is a very promising drug for anti-rejection therapy. So far there were no r e ports on its anti-rejection effects on animal organ transplantation. T hi s paper furnished evidence of its value in that respect. Our results demonstrated that CS-1 of a given concentration possesses d i s tinct anti-rejection effects. Considering the mechanism of rejection in organ transplantation, we postulate that the anti-rejection effects of CS-1 might be related to i t s i n h i b i t o r y effects on helper T cells and enhancing effects on suppressor T cells. Wang et al ~a~ reported that CS-1 could i n h i b i t T lymphocyte blastogenesis of spleen lymphocytes from different strains of mice b y ConA and PHA, markedly enhance the function of suppressor T cells induced by ConA in vi t r o and apparently enhance the function of spontaneous suppressor T lymphocytes in v i v o . These findings support our postula-
tion. Our experimental results also s u g gested that CS-1 exerted no detrimental effects o n spleen and thymus of rats, and it did not demonstrate h e p a t o t o x i city and nephrotoxicity. In addition, it could effectively prevent rejection and prolong the s u r v i v a l of grafts without causing infection. All these findings suggested that the anti-rejection effects of CS-1 are selective. The good anti-rejection effects of CS-1 might be related to its induction of immuno-tolerance. According to l i terature t~ the allografts of rats or mice were believed to develop immuno-tolerance if the s u r v i v a l time exceeded 60 days. In our experiment, 4 rats (30o~) survived over 90 days in 4o~ CS-1 group. We hold that the 4 rats
did develop immuno-tolerance to heart allografts. It is currently believed that suppressor T cells play an i m p o r tant role in the formation and maintenance of immunological tolerance. After their activation, they can produce a soluble i nhi bi t or of immune reaction, thereby i nhi bi t i ng function of the helper T cells and immune response of the body. CS-1 might induce i m m u n o tolerance by enhancing the function of the suppressor T cells. The anti-rejection effects of CS-1 apparently depended on the drug concentration: the effects of ~ CS-1 were obvi ousl y greater than those of 1 6 ~ CS-1. T hi s may be attributed to the dual effects of the immunosuppressant, the mechanism of which still awaits further investigation. From our experimental results, we are led to conclude that CS-1 is a new very promising immunosuppressant or immuno-regulator which might be a p plied in clinical allotransplantation in the future.
REFERENCES 1. One K, et al. Improved technique of heart transplantation in rat. J Thorac Cardiovasc Surg 1969;57:225"7. 2. Lee S, et al. A simplified model for hoterotopic rat heart transplantation. Transplanlation. 19~2~33:438-40. 3. T ~ , ~ , & ~ ~ l ~ . 4. ~,~,
~.
~:~z_.~_~r
,~,,~ 1984;9(4):248-50. S . ~,"~"~, ~ . ~ t ~ r IV. ~ ~ ~,~.~- ~ ~ ~ ~ , ] ~ ~r~ ~.. ~]~~ ' ~. ~ ~ ~[~ 19g@;17(5): 329-31. 6. ~&, ~. ~ ~ ~ 2 . v. 1988:17 (5) : 332-4. 9 Mayumi H, et al. Drug-induced tolerance to allografts in mice.- V. Prolongation of skirt graft survival in tolerant mice with combined immunosuppressive treatments. Transplantation 1985)39,$35-8.
