| Objective:To explore the effect of echinacoside on improving spermatogenic dysfunction and its related mechanisms in rats,and to provide theoretical basis for the application of echinacoside in promoting spermatogenic function.Methods:In vivo experiment,60 Wistar male adult rats were fed normally for 5 days and randomly divided into 6 groups(10 rats in each group:normal group,model group,positive control group,25mg/kg echinoside dose group(ECH L group),50mg/kg echinoside dose group(ECH M group),and 100mg/kg echinoside dose group(ECH H group)after weighing.Except for the normal group,the rats in the other groups were intraperitoneally injected with lead acetate for 7 consecutive days,weighed every 3 days,and administered according to body weight(the dose was 25mg/kg)to prepare the model of lead acetate poisoning.After successful modeling,the positive drug group received intraperitoneal injection of vitamin C 10mg/Kg·d,echinacoside 25 mg/kg,echinacoside 50 mg/kg and echinacoside 100 mg/kg were given to the low,medium and high echinacoside groups respectively,and the model group was given the same volume of normal saline for 30 consecutive days.Twenty-four hours after the last gavage,rats were anesthetized with 10%chloral hydrate(The rats were injected with 0.3mL/100g dose according to their body weight),and epididymis was taken to detect sperm survival rate,motility and deformity rate.The contents of SOD,LDH and GSH were measured in the left testis.Western blot(WB)was used to detect the expression changes of key proteins(including JNK,p-JNK,p38,p-p38)that activate MAPK signaling pathway in testicular tissue.HE staining was performed on the right testis of rats to observe the histological changes of the testis.In vitro experiment,400 mol/L hydrogen peroxide was applied to mouse testicular mesenchymal cells(TM3)for 4 hours to prepare the cell model of hydrogen peroxide damage.The final concentrations of echinoside in cell culture medium were 50 mol/L,100 mol/L and 200 mol/L,respectively.Low-dose,medium-dose and high-dose groups of echinoside were set up,and the effects of different concentrations of echinoside on TM3 activity were detected by MTT method 24 hours after echinoside was applied to TM3 cells.LDH,GSH-PX and T-AOC kit were used to detect the changes of oxidative stress indicators in TM3 cells under the action of echinolin at different concentrations,and WB detected the expression changes of key proteins that activate MAPK signaling pathway in TM3(including JNK,P-JNK,P38 and P-P38).The changes of protein expression in cells after echinoside treatment were analyzed.Results:In vivo experimental results showed that compared with the normal group.the sperm survival rate,motility and deformity rate in the model group decreased(p<0.05),SOD activity decreased(p<0.05),LDH content decreased(p<0.05).GSH content decreased(p<0.01).In WB detection of expression of key proteins JNK and p-38 protein of MAPK signal pathway in testis tissue,Compared with the normal group,the phosphorylation expression of JNK and p-38 protein in the model group increased significantly(p<0.01).Compared with the model group,the positive control group(VC group)has increased sperm survival rate and motility(p<0.05),decreased deformity rate(p<0.01),and increased SOD activity,LDH content and GSH content in testis tissue,but there is no significant difference(p>0.05).The phosphorylation expression of JNK and p-38 protein detected by WB decreased significantly(p<0.05).Compared with the model group,the survival rate and activity of sperm in the low and medium dose groups of echinacoside increased(p<0.05),SOD activity and LDH content in testis tissue increased,but there was no significant difference(p>0.05).The phosphorylation expression of p-38 protein detected by WB in echinacoside low dose group decreased,but there was no significant difference(p>0.05),and the phosphorylation expression of JNK protein decreased significantly(p<0.05).The deformity rate was decreased(p<0.01)and GSH content was significantly increased(p<0.01)in echinacoside medium dose group.The phosphorylation expression of p-38 protein detected by WB decreased(p<0.05),and the phosphorylation expression of JNK protein decreased significantly(p<0.01).In high dose echinacoside group,sperm survival rate increased(p<0.01),sperm motility increased(p<0.01),sperm deformity rate decreased(p<0.01),LDH content in testis tissue increased,but there was no significant difference(p>0.05),SOD activity increased(p<0.01),GSH content increased(p<0.05).The phosphorylation expression of JNK and p-38 protein detected by WB decreased significantly(p<0.01).HE staining results of rat testis tissue showed that echinacoside can improve testis structural damage.The results of in vitro experiments showed that MTT determined that the optimal stimulation condition was 100?mol/L echinacoside acting on cells.The anti-oxidation ability of TM3 cells treated with echinacoside was significantly increased by kit detection.Compared with the normal group,the phosphorylation expression of JNK protein in the model group cells increased significantly(p<0.05),and the phosphorylation expression of JNK protein increased significantly(p<0.01).compared with the model group,the phosphorylation expression of JNK and p-38 protein in TM3 cells treated with echinacoside decreased significantly(p<0.01),and the cell damage gradually recovered.Conclusion:Echinacea glycoside can improve sperm quality in rats with spermatogenic dysfunction.Echinacea glycoside can reduce oxidative stress injury level of TM3 cells caused by oxidative stress injury of testis and hydrogen peroxide in rats with spermatogenic dysfunction caused by lead acetate.Echinacea glycoside can improve testicular morphology and structure in rats with spermatogenic dysfunction caused by lead acetate.Echinacea glycoside can affect the expression of p38 and JNK proteins in testis tissue and TM3 cells,and may play an anti-spermatogenic function by regulating MAPK signaling pathway. |
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