| Objective: Stable epididymal microenvironment plays an important role in sperm maturation and fertilization ability.Varicocele can cause the reduction of antioxidants and inflammation in the epididymal microenvironment,resulting in changes the level of cytokines in the epididymis,which is important to study the effect of varicocele on male infertility.The purpose of this study was to observe the regulation of modified Da Huang Zhe Chong granules on the levels of IL-2,IL-6 and TNF-α in Testes of Varicocele Model Rats Based on Epididymal Microenvironment,providing a new idea for the study of the mechanism of male infertility caused by varicocele.Method: 40 young clean grade adolescent male SD rats were randomly divided into the sham operation group(n=10),the model group(n=10),the Lavocarnitine group(n=10)and the modified Da Huang Zhe Chong granules group(n=10).The rats in the sham operation group only opened the abdominal cavity without any treatment,and the rats in the other three groups were made varicocele model according to Turner partial left renal vein constriction.The rats in the VC group and the sham operation group were given 0.9% normal saline.The Levocarnitine group were given 0.1mg/m L levocarnitine in 20 m L 0.9% normal saline.The modified Da Huang Zhe Chong granules group was given 1.2g/m L containing Da Huang Zhe Chong water granules.The above groups were given intragastric administration volume of 1m L/100 g once a day for 8 weeks.After 8 weeks of administration,the rats in each group were weighed and killed by severing their necks.The bilateral testis and epididymis tissues were removed by laparotomy.The sperm quality of epididymis was detected by sperm counting plate and color sperm quality analysis system.The pathological changes of local microstructure of epididymis in each group were observed by Hematoxylin Eosin staining.The pathological changes of local ultrastructure of epididymis in each group were observed by transmission electron microscope.The expression of Nrf2 and HO-1 in the left epididymis was detected by histochemical method.Fluorescence quantitative Polymerase Chain Reaction was used to detect the expression of apoptosis genes IL-2,IL-6,TNF-α,Nrf2-1 and HO-1 in epididyma l tissue of rats in each group.Result:1.The results of semen quality showed that compared with the sham operation group,the sperm density and sperm motility decreased significantly in the VC group,and the semen in the model group was significantly lower than that in the sham operation group.Compared with VC group,the sperm density and sperm motility were significantly improved in Levocarnitine group and modified Da Huang Zhe Chong granules group(P<0.05).Compared with the Levocarnitine group,there was no significant difference in sperm density in the modified Da Huang Zhe Chong granules group,and there was no statistical significance(P>0.05).2.Analysis of microstructure and ultrastructure: in the sham operation group,the epithelial cells were clear and intact,and the mitochondria and organelles were at the normal level.In the VC group,the epithelial cells were blurred,and its structure was incomplete with vacuolar degeneration appeared.There were scattered lymphocytes in the tissue.In the Levocarnitine group,the epithelial cells showed slight pathological changes,and the structural morphology of mitochondria and lysosomes were slightly different,all of which were relatively intact.A large number of mature sperm cells,epithelial cells and organelles were intact and seen in the modified Da Huang Zhe Chong granules group but did not return to the normal level.3.Immunohistochemical detection: the positive expression of Nrf2/HO-1 in the left epididymis was obvious in the sham operation group,the cell luminance was low and the positive expression of Nrf2/HO-1 in the left epididymis was scattered in the VC group,and the luminance of the positive cells in the Jiawei Dahuang Zhechong formula group was significantly higher than that in the left carnitine group and VC group.4.The results of fluorescence quantitative PCR showed that:(1)IL-2: Compared with the sham operation group,the expression of gene IL-2 in epididymal cells was significantly increased in the model group(P<0.01).Compared with the model group,the content of IL-2 was decreased significantly in the levocarnitine group and the modified Da Huang Zhe Chong granules group(P<0.01).Compared with the levocarnitine group,the content of IL-2 was decreased significantly in the modified Da Huang Zhe Chong granules group(P<0.01).(2)IL-6: Compared with the sham operation group,the expression of gene IL-6 in epididymal cells was significantly increased in the model group(P<0.01).Compared with the model group,the content of IL-6 was decreased in the modified Da Huang Zhe Chong granules group(P<0.01),there was no significant difference in the downregulation of IL-6 expression in the levocarnitine group(P>0.05).(3)TNF-α: Compared with the sham operation group,the expression of gene TNF-α in epididymal cells was significantly increased in the model group(P<0.01).Compared with the model group,the content of TNF-α was decreased significantly in the levocarnitine group and the modified Da Huang Zhe Chong granules group(P<0.01).Compared with the levocarnitine group,the content of TNF-α was decreased significantly in the modified Da Huang Zhe Chong granules group(P<0.01).(4)Compared with the sham operation group,the relative expression content of epididymis gene Nrf2 in the VC model group decreased significantly,compared with the VC model group,the Nrf2 content in the levocarnitine group and the Jiawei Dahuang Zhechong formula group increased significantly(P<0.0l),and compared with the levocarnitine group,the Nrf2 expression content in the Jiawei Dahuang Zhechong formula group increased significantly(P<0.0l);(5)HO-1: Compared with the artificial hand group,the relative expression content of epididymis gene HO-1 in the VC model group decreased significantly,and compared with the VC model group,the HO-1 content increased significantly in the levocarnitine group and the modified Jiawei Dahuang Zhechong formula group(P<0.01).Compared with the levocarnitine group,the Jiawei Dahuang Zhechong formula group could significantly increase the expression of Nrf2,but there was no significant difference between the two groups(P>0.05).Conclusion:In the experimental results,the local micropathological changes were significantly changed in the model VC group.Compared with the model group and levocarnitine group,the gene expression contents of IL-2,IL-6 and TNF-α in modified Da Huang Zhe Chong granules group decreased significantly,while the target gene and protein expression content of Nrf2 and HO-1 increased significantly,suggesting that modified Da Huang Zhe Chong granules can improve the structure of epididymal epithelial cells and promote the development and maturation of spermatozoa.In order to improve the quality of sperm,it is suggested that modified Da Huang Zhe Chong granules can improve the epididymal microenvironment and repair the injured tissue of epididymis,which has a certain obvious therapeutic effect on the treatment of varicocele male infertility and provides a new idea for the treatment of infertility in traditional Chinese medicine. |
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