| Research background and Objectives:In this study,We used cyclophosphamide to make rats model of oxidative stress leading to damage to BTB,and the model animals was treated by the Chinese medicine “Qiangjing Tablet” which has been confirmed to have anti-oxidation effect in our early study.Using the BTB-related proteins ZO-1,Occludin,F-actin,oxidative stress indicators SOD,MDA and p38 MAPK key protein p38 protein as indicators,we explored the target and repair mechanism of Bushen Huoxue TCM on damaged BTB,clarify the pathogenesis of infertility in Chinese medicine,and lay the foundation for prevention and treatment of infertility by Chinese medicine.Method:50 healthy male SD rats were randomly divided into blank control group(20rats,saline intraperitoneal injection)and model group(30 rats,cyclophosphamide intraperitoneal injection).After the model was completed,10 rats in each of the above two groups were randomly selected and tested for model verification.The remaining20 rats of the model group were randomly divided into 2 groups:Qiangjing tablet treatment group and the model control group,with 10 rats in each group.The Qiangjing Tablet treatment group was given an equivalent dose of 0.45g/kg·d of Chinese medicine(equivalent to 6 times of 5.25 g of crude drug/60kg·d of 1 clinical person).The model control group and the blank control group were given equal doses of normal saline,and the three groups were intragastrically once a day for 4weeks.After the experiment,the changes of semen index in rats were detected by computer-assisted semen analysis.The testicular tissue structure was observed under light microscope.The levels of SOD and MDA in testicular tissues were measured by ELISA.Immunohistochemistry S-P staining was used to detect the expression of tight junction proteins Occludin and Zo-1.The expression of cytoskeletal protein F-actin was detected by immunofluorescence staining of testis tissue.The expression of Occludin,F-actin and p38 in testis tissues was detected by Western blot.The study data was statistically analyzed using SPSS 22.0 software.Results:1.Pathological tissue structure under light microscope: After the model was completed,the spermatogenic cells of the testicular seminiferous tubules were reduced to different extents,the wall of the tube was sunken or even collapsed,the wall of the tube was thinned,the basement membrane was thickened,and the sperm in the lumen was Reduced to varying degrees;After the treatment of Qiangjing tablets,the spermatogenic cells of the seminiferous tubules of the testis increased in different degrees,the degree of wall depression was reduced to some extent,the thickness of the wall was relatively uniform,and the spermatozoa in the lumen increased to different degrees.2.The SOD of the model control group was significantly lower than that of the normal control group(P<0.05),and the MDA content was significantly higher than that of the normal control group(P<0.05).The SOD of the Qiangjing Tablet group was significantly higher than that of the control group before treatment(P<0.05),MDA.The content was significantly lower than the model control group(P<0.05)3.After the model was completed,the positive staining of Occludin and Zo-1immunohistochemistry was significantly reduced.After 4 weeks of natural recovery,the positive staining increased slightly.The positive staining of Qiangjing Tablet group was significantly higher than that of the model control group.4.After the model was completed,the positive fluorescence intensity of F-actin decreased significantly.After 4 weeks of natural recovery,the positive fluorescence intensity increased significantly.The positive fluorescence intensity of the Qiangjing Tablet group was stronger than that of the model control group.5.After the model was completed,the expression of Occludin and F-actin protein decreased significantly(P<0.05),and the expression of p38 protein increased significantly(P<0.05).After 4 weeks of natural recovery,the expression levels of Occludin and F-actin were all increased.The expression of p38 protein decreased,but the difference was not statistically significant.The expression of Occludin and F-actin protein in Qiangjing Tablet group was significantly higher than that in the model control group(both P<0.05),and the expression of p38 protein was compared withthe model.The group decreased significantly(P<0.05).Conclusion:1.Cyclophosphamide can increase MDA level,decrease SOD level,up-regulate p38 protein expression level,down-regulate Occludin,Zo-1,F-actin protein expression level,and reduce sperm number.It suggests that cyclophosphamide may increase testicular oxidative stress level,activate p38 MAPK pathway,reduce BTB-related protein expression and damage BTB.2.Qiangjing tablets can decrease MDA level,increase SOD level,down-regulate p38 protein expression level,up-regulate Occludin,Zo-1,F-actin protein expression level and increase sperm quantity,which indicates that Qiangjing tablet may restore the expression function of BTB-related protein,repair damaged BTB and improve sperm quality by reducing oxidative stress level of testis,inhibiting p38 MAPK pathway. |
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