Protective Effect Of HNG On Cyclophosphamide-induced Ovarian Injury In Rats And Its Preliminary Mechanism

Part 1 Establishment of a rat model of ovarian injury induced by cyclophosphamide[Purpose] Using the methods commonly used in the literature,the rat model of ovarian injury was established by intraperitoneal injection of CTX.[Methods] According to the model method in the published literature [18],20 female Sprague-Dawley(SD)rats of 10 weeks old,280-300 g,with normal estrous cycle were randomly divided into two groups: Control group and CTX group(ovarian injury model group).Then the drugs were injected intraperitoneally : CTX group : Rats received intraperitoneal injection of CTX 50 mg/kg on the first day,then intraperitoneal injection of8mg/kg per day for 14 consecutive days;Control group: Rats received intraperitoneal injection of saline 1ml/kg daily for 15 days.All rats were weighed and recorded before intraperitoneal injection.Vaginal smears were taken at 9: 00 a.M.every day from the beginning of administration to the end of administration,and the cell types were observed under light microscope.After administration,the weight of the rats was weighed on the second day,and the bilateral ovarian tissues were removed,one ovary was frozen in liquid nitrogen,and the other ovary was embedded in paraffin,serial sections were used for follow-up experiments.Monitoring of estrous cycle in rats by Wright’s Giemsa staining;HE staining was used to observe the morphological changes of rat ovary under microscope,and the number of follicles at all levels was calculated at the same time;Using ELISA method to detect the level of AMH in rat serum.[Result] Compared with the control group,the body weight(P<0.01),ovarian index(P<0.05)and serum AMH level(P<0.05)of rats in CTX group decreased significantly,the number of rats with estrous cycle disorder increased;At the same time,ovarian structure disorder and tissue fibrosis were found in CTX group;The number of primordial follicles(P<0.01)primary follicles(P<0.01),secondary follicles(P<0.05)and mature follicles(P<0.05)decreased significantly,while the number of atretic follicles increased(P<0.01).[Conclusions] CTX-induced SD rat ovarian injury model was successfully established.Part 2 Protective effect of HNG on ovarian injury induced by CTX in rats[Purpose] To study the protective effect of HNG on ovarian injury induced by CTX in rats.[Methods] 40 female SD rats of 10 weeks old,280-300 g,with normal estrous cycle were randomly divided into four groups after one week of feeding:Control group,CTX group,CTX+HNG group and HNG group,then the drug was given by intraperitoneal injection.All rats were weighed and recorded before intraperitoneal injection.Vaginal smears were taken at 9: 00 a.M.every day from the beginning of administration to the end of administration,and the cell types were observed under light microscope.Among them,the mode of administration was as follows: Control group: rats received intraperitoneal injection of saline 1ml/kg daily for 15 days;CTX group: rats received intraperitoneal injection of CTX 50 mg/kg on the first day,and then intraperitoneal injection of 8 mg/kg per day for 14 consecutive days;CTX+HNG group: HNG was injected intraperitoneally for one week,then CTX+HNG was given continuously for 15 days,and CTX was injected intraperitoneally three hours after HNG administration;HNG group:rats received intraperitoneal injection of 5mg/kg HNG every day for 15 days.After administration,the weight of the rats was weighed on the second day,and the bilateral ovarian tissues were removed,one ovary was frozen in liquid nitrogen,and the other ovary was embedded in paraffin.Serial sections were used for follow-up experiments.The body weight,ovarian wet weight,ovarian index,the number of rats with abnormal estrous cycle,the morphological changes of ovarian tissue,the number of follicles at all levels and the apoptosis rate of ovarian granulosa cells(Tunel staining)were measured.At the same time,the expression levels of PI3K/Akt protein,pro-apoptotic protein caspase3 and anti-apoptotic protein Bcl-2were detected by Western-Blot method.[Results] Compared with CTX group,the number of abnormal estrous cycle in CTX+HNG group decreased significantly after HNG administration;The ovarian wet weight and ovarian index of rats increased significantly(P<0.01),the weight of rats decreased but the difference was not statistically significant(P>0.05);The number of primordial follicles,primary follicles and mature follicles increased(P<0.05),while atretic follicles decreased(P<0.05),ovarian tissue fibrosis was alleviated,The number of apoptotic cells decreased significantly(P<0.01).HNG can antagonize the expression of PI3 K and AKT phosphorylated protein in ovarian tissue induced by CTX,and HNG can promote the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein cspase3 in ovarian tissue.[Conclusions] HNG has a protective effect on CTX-induced ovarian injury in rats,and HNG may inhibit CTX-induced apoptosis of ovarian granulosa cells by affecting PI3K/Akt signal pathway,caspase3 and Bcl-2 protein.Part 3 The mechanism of HNG inhibiting CTX-induced ovarian injury[Purpose] To investigate whether HNG protects rat ovarian granulosa cells from apoptosis induced by CTX through PI3K/Akt signal pathway.[Methods]In vitro cell experiment,we carried out with 4-HC(an active metabolite of CTX),COV434(a human granulosa cell line),and LY294002(an inhibitor of PI3K/Akt).After COV434 cells were treated with different concentrations of 4-HC for 24 hours,the apoptosis rate of each group was measured by flow cytometry,and the appropriate concentration was selected for further experiment.Next,COV434 cells were pretreated with different concentrations of HNG for 1 hour,and then co-incubated with4-HC for 24 hours,The apoptosis rate of each group was measured by flow cytometry,and then select the HNG concentration corresponding to the apoptosis rate that has no statistical difference between the control group and the control group.Then,COV434 cells were divided into four groups: control group,4-HC group,4-HC+HNG group and4-HC+HNG+LY294002 group(LY294002 group),the apoptosis rate of each group was measured by flow cytometry;At the same time,the expression levels of PI3 K / Akt signaling pathway protein and its phosphorylated protein,pro-apoptotic protein caspase3 and anti-apoptotic protein Bcl-2 were detected by Western blot method.[Results] 4-HC induced apoptosis in COV434 cells in a dose-dependent manner,and significantly induced apoptosis when cells were treated with 10μM 4-HC(P<0.05).HNG pretreatment could decrease the apoptosis of COV434 cells induced by 4-HC,and when the concentration of HNG was 10μg/ml,the apoptosis rate of COV434 cells was not significantly different from that of the control group(P>0.05),which indicated that HNG could reverse the apoptosis of COV434 cells induced by 4-HC at appropriate concentration.However,after adding PI3K/Akt inhibitor LY294002,there was no significant difference in apoptosis rate between 4-HC group and LY294002 group(P>0.05),indicating that LY294002 blocked the anti-apoptotic effect of HNG.Next,we evaluated the expression levels of PI3K/Akt pathway proteins and apoptosis-related proteins Bcl-2 and caspase3 among the four groups.The results showed that compared with the control group,the expression level of p-PI3 K,p-Akt and Bcl-2 protein decreased(P<0.05)and the expression level of caspase3 increased in 4-HC group.Compared with 4-HC group,the expression level of p-PI3 K,p-Akt and Bcl-2 protein in 4-HC+HNG group increased,while the expression level of caspase3 decreased.Compared with 4-HC group,there was no significant difference in p-PI3 K,p-Akt,Bcl-2 and caspase3 protein expression in LY294002 group.[Conclusions]HNG pretreatment could decrease the apoptosis of COV434 cells induced by 4-HC,but LY294002,an inhibitor of PI3K/Akt,blocked the anti-apoptotic effect of HNG.It is suggested that HNG protects C0V434 cells from 4-HC-induced apoptosis by activating PI3K/Akt signal pathway.