| Objective:To explore the molecular mechanism of XJCRTST,a traditional Chinese medicine compound for tonifying kidney,replenishing essence,nourishing blood and promoting blood circulation,through PINK 1/Parkin mediated mitochondrial autophagy pathway,resisting oxidative stress injury,repairing granulosa cell injury in ovarian hypofunction rats and human,and then promoting follicular development.Methods:(1)Triptolide(TP)was administered to SD rats(3 months old sexual maturity,weight about 220g).The rats were randomly divided into blank control group,400 μg/kg/d 30 d group,400 μg/kg/d 40 d group,500 μg/kg/d 30 d group and 500 μg/kg/d 40 d group.There were 5 groups.And there were 5 rats in each group.Vaginal cytological smear(Pap staining)was performed every day from 10 days before the end of administration.HE staining of ovarian tissue was performed in each rat in successful model group.The levels of FSH,E2and AMH in serum of abdominal aorta were detected by Elisa method;The levels of MDA and SOD in ovarian tissue were detected by Elisa;The mitochondrial structure and mitochondrial autophagy in ovarian granulosa cells of rats in each group were observed by transmission electron microscope.(2)Thirty-six SD rats,3-month-old sexually mature female rats weighing about 220 g,were selected as experimental animals.All these rats had normal estrous cycle after screening by vaginal cytological smear.According to random number table method to enroll 6 rats in blank control group(Control),giving saline lavage daily,the rest of the 30 rats were given intragastric administration for 30 consecutive days with the TP 500μg/kg.d(dissolved in saline solution containing 5%DMSO)according to preliminary experimental results of 500 μg/kg/d,30 d group),successfully modelled SD rats were randomly divided into 5 groups.And there were 6 rats in each group.① TP model group(TP);Western medicine group(TP+EV);③ High dose of Cistanche Tusi Decoction Group(TP+XJCRTST-High);④Middle dose of Cistanche Tusi Decoction Group(TP+XJCRTST-Middle);⑤Low Dose of Cistanche Tusi Decoction Group(TP+XJCRTST-Low).The contents of E2,AMH,FSH and LH in serum were determined by ELISA after 24 hours of intragastric administration.The contents of MDA,SOD and ATP in granulosa cells were detected by ELISA,and the contents of ROS in granulosa cells were detected by flow cytometry.After weighing,the animals were killed,the complete bilateral ovaries were taken out by laparotomy,and HE staining was performed.The morphological changes of rats’ ovaries were observed under light microscope,and the primary follicles,secondary follicles and atresia follicles were counted.The number and morphology of mitochondria and mitochondrial autophagy in ovarian granulosa cells were observed by transmission electron microscope.The mitochondrial membrane potential in ovarian granulosa cells was detected by JC-1 method;Apoptosis rate of granulosa cells was detected by flow cytometry.The expression of LC3-II/LC3-I,p62,Hsp60,PINK1,Parkin in ovarian granulosa cells were detected by western blotting;The expression of LC3-II/LC3-I,p62,Hsp60,PINK1,Parkin mRNA in ovarian tissue was detected by RT-PCR.(3)Human ovarian granulosa cells(GCs)CP-H192 were purchased,and these GCs were cultured in vitro and divided into 8 groups:①blank control group(Control);TP model Group(TP);③TP+Western Medicine Group(TP+EV);④TP+high dose of drug-containing serum group(TP+XJCRTST);⑤TP+high dose of drug-containing serum+si-Pinkl group(TP+XJCRTST+si-Pinkl);⑥TP+high dose of serum containing drugs+ov-Pinkl group(TP+XJCRTST+ov-Pinkl)⑦TP+si-Pinkl group;⑧TP+ov-Pinkl group.At the same time,drug-containing serum was prepared:16 mature female SD rats aged 3 months and weighing about 220g were randomly divided into 4 groups,with 4 rats in each group.They are:①normal control group;②TP model group;③Western medicine control group;④High dose of traditional Chinese medicine group.At 37℃ and 5%CO2 saturated humidity for 48 hours,and then the culture supernatant and cells were taken for subsequent detection of various indexes:the GCs activity was detected by CCK8 method;the levels of E2,AMH,P,FSH and LH in GCs supernatant were detected by ELISA;the levels of ROS,MDA and SOD in GCs were detected by ELISA;the mitochondrial morphology and mitochondrial autophagy in GCs were observed by transmission electron microscope;the mitochondrial membrane potential in GCs was detected by JC-1 method;the apoptosis rate of GCs was detected by Annexin-FITC/PI double staining;the expressions of LC3-Ⅱ/LC3-Ⅰ,P62,Hsp60,PINK1 and Parkin in GCs were detected by Western blotting.Results:(1)Compared with rats in the blank control group,the serum AMH and E2 levels in rats in 400 μg/kg/d 30 d group,400 μg/kg/d 40 d group,500 μg/kg/d 30 d group and 500 μg/kg/d 40 d group decreased(P<0.05),while the serum FSH level increased(P<0.05);HE staining of ovarian tissue showed that the number of follicles decreased and the number of atresia follicles increased.Pap staining suggested the disorder of estrous cycle,and the results showed that the ovarian dysfunction model was established successfully,the activity of SOD decreased(P<0.05),the level of MDA increased(P<0.05),the structure of mitochondria damaged in different degrees,the number of mitochondria decreased,the structure of mitochondria cristae was blurred or even disappeared,vacuolation appeared,and the autophagy of mitochondria increased.These changes were proportional to the dose and time of TP administration.The autophagy degree of mitochondria in granulosa cells ranged from 500-40 d to 500-30 d>400-40 d>400-30 d.(2)HE staining and follicle count of rat ovary showed that compared with rats in the control group,the pathological changes of ovarian tissue of rats in TP group were significantly aggravated,including the decrease of follicles,the increase of atresia follicles,the necrosis and shedding of granular follicle cells,the cystic dilatation of follicles and the congestion of blood vessels.Compared with rats in TP group,rats in TP+XJCRTST-Low group had slightly less pathological changes,TP+XJCRTSTMiddle group had less pathological changes,TP+XJCRTST-High group had the lightest pathological changes.Results of follicle count showed that compared with rats in control group,the number of primary follicles and secondary follicles in rats in TP Group decreased significantly,while the number of atresia follicles increased significantly(*P<0.05);Compared with rats in TP group,the number of primary follicles and secondary follicles in rats in TP+XJCRTST-High group increased significantly,while the number of atresia follicles decreased significantly(#P<0.05);Compared with rats in TP+XJCRTST-High Group,rats in TP+XJCRTST-Low Group had fewer primary and secondary follicles(&P<0.05).Changes of serum sex hormones in rats showed that compared with rats control group,serum E2 and AMH levels in rats in TP Group decreased significantly,while serum levels of FSH and LH increased significantly(*P<0.05);compared with rats in TP group,serum E2 and AMH levels in rats in TP+XJCRTST-High group increased significantly,while FSH and LH levels decreased significantly(#P<0.05);compared with rats in TP+XJCRTST-High Group,rats in TP+XJCRTST-Low Group had significantly lower levels of E2 and higher levels of LH(&P<0.05).Oxidative stress and apoptosis rate of ovarian granulosa cells showed that compared with cells in control group,the levels of SOD and ATP in TP Group decreased significantly,while the levels of MDA and ROS increased significantly(*P<0.05);compared with rats in TP Group,rats in TP+XJCRTST-High Group had significantly increased levels of SOD and ATP,but significantly decreased levels of MDA and ROS(#P<0.05);compared with TP+XJCRTST-High Group,rats in TP+XJCRTST-Low Group had the lowest level of SOD(&P<0.05).Compared with rats in the control group,the apoptosis rate in rats in TP Group was significantly higher(*P<0.05);compared with rats in TP Group,the apoptosis level of granulosa cells in rats in TP+XJCRTST-High Group and TP+XJCRTST-Middle Group decreased significantly(#P<0.05);compared with rats in TP+XJCRTST-High group,rats in TP+XJCRTST-Low group had significantly higher apoptosis rate of granulosa cells(&P<0.05).Changes of mitochondria number,morphology and membrane potential(MMP)in ovarian granulosa cells showed that rats in TP group,the number of mitochondria in ovarian granulosa cells decreased,and a large number of swollen mitochondria were found,with cristae breaking,dissolving or even disappearing,and mitochondrial autophagy occasionally occurred.In rats in XJCRTST group,mitochondrial swelling decreased,and the level of mitochondrial autophagy was proportional to the dosage.Results of MMP showed that compared with rats in control group,the level of MMP in granulosa cells of rats in TP Group decreased significantly(*P<0.05),and the level of MMP in granulosa cells of rats in TP+XJCRTST-High Group and TP+XJCRTSTMiddle Group increased significantly(#P<0.05);compared with rats in TP+XJCRTST-High Group,the level of MMP in granulosa cells of rats in TP+XJCRTSTLow Group decreased significantly(&P<0.05)WB and RT-PCR indicated that compared with rats in control group,the expression of LC3-Ⅱ/LC3-Ⅰ,Hsp60,Pink1,Parkin protein and mRNA in rats in TP group increased significantly,while the expression of P62 protein and mRNA decreased significantly(*p<0.05).Compared with rats in TP group,the expression of LC3-Ⅱ/LC3-Ⅰ,Pink1,Parkin protein and mRNA in rats in TP+XJCRTST-High and TP+XJCRTST-Middle groups increased significantly,while the expression of P62,Hsp60 protein and mRNA decreased significantly(#p<0.05);Compared with rats in TP+XJCRTST-High group,the expression levels of LC3-Ⅱ/LC3-Ⅰ,PINK1,Parkin protein and mRNA in TP+XJCRTST-Low group significantly decreased,while the expression levels of P62,Hsp60 protein and mRNA significantly increased(&p<0.05).(3)Changes of GCs activity indicated that compared with control group,GCs activity in rats in TP Group decreased significantly(*P<0.05);compared with rats in TP group,GCs activity in rats in TP+XJCRTST group increased significantly(#P<0.05),GCs activity in rats in TP+XJCRTST+si-PINK1 and TP+si-PINK1 groups decreased significantly(#P<0.05),GCs activity in rats in TP+XJCRTST+ov-PINK1 and TP+ov-PINK1 groups increased significantly(#P<0.05).Changes of sex hormones in GCs supernatant showed that compared with rats in control group,the levels of E2,AMH and P in GCs of rats in TP group decreased significantly(*P<0.05);Compared with rats in TP Group,the levels of E2,AMH and P in GCs of rats in TP+XJCRTST Group significantly increased(#P<0.05),and the levels of E2,AMH and P in GCs of rats in TP+XJCRTST+ov-PINK1 Group and TP+ov-PINK1 Group significantly increased(#P<0.05);compared with rats in TP+XJCRTST group,the levels of E2,AMH and P in GCs of rats in TP+si-PINK 1 group decreased significantly(&P<0.05).Changes of oxidative stress indexes in GCs showed that compared with rats in control group,SOD content in GCs of rats in TP Group decreased significantly,MDA content increased significantly(*P<0.05);Compared with rats in TP group,the content of SOD in GCs of rats in TP+XJCRTST group increased significantly,but the content of MDA decreased significantly(#p<0.05),while the content of SOD increased significantly,but the content of MDA decreased significantly in rats in TP+XJCRTST+ov-PINK1 and TP+ov-PINK1 groups(#p<0.05);Compared with rats in TP+XJCRTST group,the content of SOD in GCs of rats in TP+XJCRTST+si-PINK1 group and TP+si-PINK1 group decreased significantly and the content of MDA increased significantly(&p<0.05).Changes of ROS level and apoptosis rate in GCs showed that compared with rats in control group,ROS level and apoptosis rate in GCs of rats in TP Group increased significantly(*P<0.05);Compared with rats in TP Group,ROS level and apoptosis rate in GCs of rats in TP+XJCRTST Group decreased significantly(#P<0.05);The ROS level and apoptosis rate in GCs of rats in TP+XJCRTST+si-PINK1 group and TP+siPINK1 group increased significantly(#P<0.05);the ROS level and apoptosis rate of GCs in rats in TP+XJCRTST+ov-PINK1 and TP+ov-PINK1 groups were significantly decreased(#P<0.05).Compared with rats in TP+XJCRTST group,rats in TP+XJCRTST+si-PINK1 group and TP+si-PINK1 group had higher level of ROS and apoptosis rate in GCs(&p<0.05).Changes of mitochondrial membrane potential(MMP)in GCs showed that compared with rats in the control group,MMP in rats in TP group decreased significantly(*P<0.05);compared with rats in TP Group,MMP in GCs of rats in TP+XJCRTST Group increased significantly(#P<0.05);MMP in GCs of rats in TP+XJCRTST+si-PINK1 group and TP+si-PINK1 group decreased significantly(#P<0.05);MMP in GCs of rats in TP+XJCRTST+ov-PINK1 and TP+ov-PINK1 groups increased significantly(#P<0.05).Compared with rats in TP+XJCRTST group,MMP in GCs of rats in TP+XJCRTST+si-PINK1 group and TP+si-PINK1 group decreased significantly(&P<0.05).Electron microscope pictures showed that the number of mitochondria in GCs of rats in TP+XJCRTST group increased,and mitochondrial autophagy was slightly more than that of rats in TP group.There was more mitochondrial autophagy in rats in TP+X J CRT S T+ov-PINK 1 group,less mitochondrial autophagy in rats in TP+XJCRTST+si-PINK1 group,more mitochondrial autophagy in rats in TP+ovPINK1 group and less mitochondrial autophagy in rats in TP+si-PINK1 group.The results of WB showed that the expression of Hsp60,PINK1,Parkin,LC3-II/LC3I in GCs of rats in TP group was significantly higher than that in control group(*p<0.05),and the expression of P62 protein was significantly lower than that in Control group(*p<0.05);Compared with rats in TP group,the expression of PINK1 and Parkin in GCs of rats in TP+XJCRTST group increased significantly(#P<0.05),the expression of LC3-II/LC3-I,the expression of Hsp60 and P62 decreased significantly(#P<0.05);the expression of LC3-II/LC3-I,PINK1 and Parkin in GCs of rats in TP+XJCRTST+ov-PINK1 and TP+ov-PINK1 groups increased significantly(#p<0.05),while the expression of Hsp60 and P62 decreased significantly(#p<0.05).Compared with rats in TP+XJCRTST+si-PINK1 and TP+si-PINK1 groups,the expression of PINK1,Parkin,LC3-II/LC3-I protein decreased and the expression of Hsp60,P62 protein increased significantly(&p<0.05)in GCs of rats in TP+XJCRTST+si-PINK1 group and TP+si-PINK1 group compared with TP+XJCRTST+si-PINK1 group.Conclusions:(1)Rat models of ovarian hypofunction were successfully established in 400 μg/kg/d 30 d group,400 μg/kg/d 40 d group,500 μg/kg/d 30 d group and 500 μg/kg/d 40 d group by intragastric administration of TP,and different degrees of ovarian injury occurred according to the dosage and time of administration.With the aggravation of oxidative stress injury,the degree of mitochondrial autophagy gradually increased,but after reaching a certain degree,it would not continue to increase with the increase of TP measurement and time.(2)Xinjia Cistanche Tusi Decoction(XJCRTST)can alleviate oxidative stress injury,mitochondrial dysfunction and mitochondrial autophagy failure induced by TP in rat and human ovarian granulosa cells;The newly added Xinjia Cistanche Tusi Decoction(XJCRTST)may protect the ovarian function of SD rats and the homeostasis of GCs in human ovary by activating mitochondrial autophagy mediated by PINK 1/Parkin to resist oxidative stress,repair granulosa cell injury and inhibit granulosa cell apoptosis. |
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