| Objective:The purpose of this experiment was to observe the effect of Yishen Tongluo recipe on sperm DNA damage in male rats exposed to benzo(a)pyrene(BaP),and to screen the differential expression of miRNA and XRCC1 protein in rats with sperm DNA damage induced by BaP,to explore the protective mechanism of traditional Chinese medicine against DNA damage in spermatogenic cells,and to provide theoretical support for traditional Chinese medicine to protect against genetic material damage caused by environmental pollutants.Methods:1.The rats were randomLy divided into 3 groups by SAS software:control group(n=10),BaP poisoning group(n=20)and Yishen Tongluo prescription group(n=20).The benzo(a)pyrene solid was diluted into the working solution of 0.1mg/mL with corn oil and 0.5%dimethyl sulfoxide(DMSO).Each rat in the BaP exposure group and Yishen Tongluo prescription group was injected intraperitoneally according to 0.1mL/100g once every day for 60 days.The procedure of intraperitoneal injection of 0.5%DMSO,at 1 mL/Kg in the control group was the same as that in the BaP group.Yishen Tongluo recipe was given intragastrically for 30 days from the 31st day after BaP exposure while the control group and BaP group were given normal saline at the same time until the end of the experiment..Finally,the samples were used for the DFI and pathological morphology of rats in each group.2.After the intervention,the frozen epididymis samples of the control group,BaP exposure group and Yishen Tongluo recipe group were ground and homogenized,and the total protein was extracted,then the protein sample was denatured and electrophoretic,gel membrane was transferred and detected.Finally,the expression results of XRCC1 protein in each group were compared.3.The total RNA,of epididymis tissue samples of rats in control group,BaP poisoning group and Yishen Tongluo recipe group were extracted.The epididymis tissue was sequenced by chip hybridization technique to obtain miRNA differential expression lineage.miRNA differential genes were screened in each control group,the differential miRNA expression was detected and the differential miRNA was analyzed by gene ontology enrichment analysis.Finally,the miR-466c,with significant differential expression was selected to verify the expression content by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR).Results:1.Compared with the control group,the DFI index in the BaP group was significantly higher(P<0.05),and the DFI in the Yishen Tongluo recipe group was lower than that in the BaP group(P<0.05).The testicular pathological sections of the control group showed that the seminiferous tubules were intact and neatly arranged.The number of seminiferous epithelium is moderate,the level is clear,mature sperm can be seen in the lumen,and the morphology of intercellular cells is not abnormal.In the BaP group,the seminiferous tubules of testicular tissue were reduced,the number of seminiferous epithelium was less,the arrangement of spermatogenic cells was loose,and the germ cells and interstitial cells were partially degenerated.Compared with the B aP group,the testicular tissue of the Yishen Tongluo recipe group was less damaged,the boundary of seminiferous tubules was still clear,the thickness of seminiferous epithelium was restored,the number of spermatogenic cells was larger,mature spermatozoa could be seen in part of the lumen,and the interstitial cells had no degeneration.2.Compared with the control group,the testicular XRCC1 protein content in the BaP exposed group was significantly lower than that in the Yishen Tongluo recipe group,and the testicular XRCC1 protein content in the BaP exposed group was significantly lower than that in the Yishen Tongluo recipe group.3.PCA and Thermographic analysis showed that there were significant differences in miRNA expression among control group,BaP exposure group and Yishen Tongluo recipe group.Compared with Yishen Tongluo recipe group,43 differentially expressed miRNA were up-regulated and 22 down-regulated,while compared with BaP-exposed group,the differential expression of miRNA in control group was 13 up-regulated and 16 down-regulated.Compared with BaP group,the differential expression of miRNAin Yishen Tongluo recipe group was 39 up-regulated and 20 down-regulated.Functional enrichment analysis showed that these differences were mainly concentrated in cell process,cell composition,cell adhesion and catalytic activity.Among these differential genes,we found that the difference multiple of miR-466c changed significantly,and the expression was significantly down-regulated in the BaP expo sure group.After the intervention of Yishen Tongluo recipe,the difference multiple of miR-466c level changed and up-regulated significantly.Conclusions:After rat sperm DNA damage induced by Bap,Yishen Tongluo recipe can improve the degree of testicular tissue injury,promote the repair of rat germ cells by up-regulating the expression of miR-466c and XRCC1 protein,reverse the process of sperm DNA damage caused by Bap poisoning,and improve the integrity of sperm DNA. |
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