| ObjectiveBased on miR-146a/23a regulating TLR2/4/NF-κB/PD-L1 and STAT3/PD-L1 signaling pathway as the target,and to explore the expressions of related proteins in the pro-inflammatory signaling pathway TLR2/4/NF-κB and STAT3 and the changes on the anti-inflammatory molecules of miR-146a,miR-23a and PD-L1 in the 43℃ testicular heat stress model through bioinformatics,animal and cell experiments.In addition,it’s to investigate the effects of Wuzi Yanzong pill(WYP)on pro-inflammatory and anti-inflammatory signals in testis,so as to restore the local immune balance of testis and provide a favorable immune microenvironment for spermatogenesis.At the same time,it provides experimental basis for WYP to treat male infertility caused by heat stress.Methods1.BioinformaticsThe target genes of miR-146a-5p and miR-23a-3p were predicted and analyzed by bioinformatics.The target genes of miR-146a-5p and miR-23a-3p were downloaded from TargetScan,miRDB and miRWalk bioinformatics websites,respectively,and Venn was used to obtain the intersection of the target genes of the three websites,and a total union genes were obtained by combining the above two intersection genes with the target genes supplemented by literature search.Next,submitting the total union genes into STRING 11.0 database to get the analysis data which then were imported into CytoScape 3.7.1 to construct PPI network,and then proceeding the network topology analysis to obtain the key target genes for miR-146a-5p and miR-23a-3p.At last,the key target genes were enrichment analysed and visualized by GO and KEGG in DAVID database.2.Animal experiments(1)The changes of miR-146a-5p,miR-23a-3p and TLR2/4/NF-κB and STAT3 signaling pathways and the regulatory effects of Wuzi Yanzong pill(WYP)in heat-stress testicular tissues were detected.35 male SD rats were randomly divided into 5 groups,including Control group,Model group,Wuzi Yanzong Pills low-dose group(WYP-L),Wuzi Yanzong Pills medium-dose group(WYP-M)and Wuzi Yanzong pills high-dose group(WYP-H),with 7 rats in each group.Administration:According to the gavage volume of 1 mL/100g,WYP-L,WYP-M and WYP-H groups were given Wuzi Yanzong pills medicine liquid with dosages of 0.5 g/kg/d,1 g/kg/d,2 g/kg/d,and the normal and model groups were given the same volume of normal saline,continuously for 15 days.Modeling:At the 11th day,the normal groups were placed at room temperature,while the model group.WYP-L.WYP-M and WYP-H groups were subjected to 43℃ heat stress modeling.30 min each time and continued 5 consecutive days.At the end of modeling,bilateral testicles were taken.The gene expressions of miR-146a-5p and miR-23a-3p in rat testis were detected by RT-qPCR.The expressions and localization of testicular tissue proteins were detected by immunohistochemistry.Western blotting assay was used to detect the proteins’ changes of the TLR2/4/NF-κB and STAT3 signaling pathway.(2)The relationship between NF-κB and PD-L1 was investigated by intraperitoneal injection of NF-κB inhibitor in rats.20 male SD rats were randomly divided into Control group,Model group NF-κB inhibitor group(PDTC)and WYP-H group,with 5 rats in each group and continuously for 15 days.Modeling:At the 11th day,the normal groups were placed at room temperature,while the model group.NF-κB inhibitor group and WYP-H group were subjected to 43℃ heat stress,30 min each time and continued 5 consecutive days.but differently,NF-κB inhibitor group were received intraperitoneal injection of NF-κB inhibitor(PDTC)2 h before heat stress(100 mg/kg).Taken bilateral testicles at the end of modeling.The expressions of PD-L1 were measured by Western blotting assay.3.Cell experiments(1)The inhibitory effects of miR-146a-5p and miR-23a-3p on target genes were verified by overexpression of miR-146a-5p and miR-23a-3p mimics.Testicular primary Sertoli cells were randomly divided into 4 groups,including Control group,TransMate group and NC mimics group in common,and respectively in miR-146a-5p mimics group and miR-23a-3p mimics group.The expressions and localization of Vimentin and WT1 proteins were detected by immunofluorescence to evaluate the purity of Sertoli cells.The survival rates of Sertoli cells transfected with different concentrations of miRNA(2.5 nM.5 nM,10 nM,20 nM and 30 nM)were determined by CCK-8 assay.Transfection efficiency of miRNA with different concentrations was detected by FAM fluorescence labeling.The gene expressions of miR-146a-5p and miR-23a-3p in Sertoli cells were verified by RT-qPCR assay.Western blotting was used to verify the TLR2/4/NF-κB and STAT3 signaling pathway proteins inhibited by miR-146a-5p and miR-23a-3p mimics.(2)The changes of miR-146a-5p,miR-23a-3p and TLR2/4/NF-κB and STAT3 signaling pathways and the regulatory effects of Wuzi Yanzong pill(WYP)in heat-stress Sertoli cells were detected.The mechanisms of Sertoli cells under heat stress:Testicular primary Sertoli cells were randomly divided into 5 groups,including Control group,Model group,Wuzi Yanzong Pills low-dose group(WYP-L),Wuzi Yanzong Pills medium-dose group(WYP-M)and Wuzi Yanzong pills high-dose group(WYP-H).Administration:The concentrations of WYP-L,WYP-M and WYP-H were 5 mg/mL.10 mg/mL and 15 mg/mL.respectively,and the Control and Model group were given the same volume of sterile PBS.Modeling:After 24 h,except for the normal group,the other four groups were subjected to 43℃ heat stress treatment for 30 min.After modeling,cells were put back into the CO2 incubator for further cultured for 24 h,and followed by molecular biology experiments.The gene expressions of miR-146a-5p and miR-23a-3p in Sertoli cells were detected by RT-qPCR.The expressions and localization of Sertoli cells proteins were detected by immunohistochemistry.Western blotting assay was used to detect the proteins’ changes of the TLR2/4/NF-κB and STAT3 signaling pathway.Results1.BioinformaticsThe prediction results and functional analysis of target genes of miR-146a-5p and miR-23a-3p.A total of 591 target genes of miR-146a-5p and miR-23a-3p were predicted through 3 miRNA databases and literature supplement.The 22 key target genes were screened out by CytoScape 3.7.1 PPI network analysis,including CREBBP,SMAD3,TRAF6,NCOA1,TLR4,UBE2D3.IRAK1,STAT5B,NFKB1,and TLR2.GO analysis showed that these key target genes were significantly enriched in the positive and negative regulation of RNA polymerase Ⅱ promoter transcription,Toll-like receptor signaling pathway,I-kappaB kinase/NF-κB signaling,innate immunity,positive regulation of type Ⅰ interferon production,and positive regulation of inflammatory response(P<0.01).KEGG analysis showed that these key target genes were significantly enriched in toll-like receptor signaling pathway(TLRs),nuclear factor κB(NF-κB)signaling pathway and mitogen activated protein kinase(MAPK)signaling pathway(P<0.01).2.Animal experiments(1)Regulation of miR-146a-5p and miR-23a-3p and TLR2/4/NF-κB and STAT3 signaling pathways in heat-stress testis by Wuzi yanzong Pill(WYP).qPCR results showed that the expression levels of miR-146a-5p and miR-23a-3p in testis of model group were significantly increased after heat stress(P<0.05).Immunohistochemistry showed that the positive expression of TLR2,TLR4,p-IRAK1,p-ERK1/2,p-STAT3 and PD-L1 protein in model group were significantly higher than those of normal group,and the protein expression locations were different at different stages of spermatogenesis.Western blotting showed that the expression levels of TLR2,TLR4,p-IRAK1(Phospho T100),p-ERK1/2(Phospho T202 and Phospho T185),and p-STAT3(Phospho TS727),p-p65 and PD-L1 were significantly increased in the model group(P<0.05).increasing in protein phosphorylation.WYP not only decreased the expression of the pro-inflammatory signaling pathway TLR2/4/NF-κB and STAT3,but also decreased the expression levels of anti-inflammatory signaling of miR-146a-5p and miR-23a-3p and PD-L1 proteins(P<0.05),and repaired testicular tissue damage caused by heat stress.(2)Effects of NF-κB inhibitor on PD-L1 expression in heat-stress testis.Compared to the normal group,it was significantly increased of PD-L1 expression in model group(P<0.05).Compared with the model group,PD-L1 expression was also inhibited by NF-κB inhibitor even after heat stress(P<0.05),and the WYP-H group were also obviously decreased(P<0.05).3.Cell experiments(1)Inhibitory effect of overexpressed transfection of miR-146a-5p and miR-23a-3p on target genes.Immunofluorescence showed that Vimentin and WT1 were expressed in cytoplasm and nucleus respectively,and the fluorescence expression rate was above 95%,which indicated that it obtained the highly purity of Sertoli cells.CCK-8 and FAM fluorescence labeling experiments showed that it could get the best survival rate and fluorescence intensity at the concentration of 10 nM miR-146a-5p and(>80%),which could be used for subsequent transfection experiments.RT-qPCR results showed that the expression level of miR-146a-5p mimics after transfection was about 1000 times than the negative control group(NC),and the expression level of miR-23a-3p mimics was more than 300 times,all with significant differences(P<0.05).Western blotting showed that overexpression of miR-146a-5p and miR-23a-3p significantly inhibited the expression of TLR4.p-IRAK1.p-p65 and PD-L1(P<0.05),but there was no statistical significance in the inhibition of TLR2(P>0.05).miR-23a-3p significantly inhibited p-STAT3(P<0.05),but miR-146a-5p had no statistical significance on p-STAT3 inhibition(P>0.05).(2)Regulation of miR-146a-5p and miR-23a-3p and TLR2/4/NF-κB and STAT3 signaling pathways in heat-stress testis by Wuzi yanzong Pill(WYP).RT-qPCR results showed that the expression levels of miR-146a-5p and miR-23a-3p were significantly increased in heat-stress Sertoli cells(P<0.05).Immunofluorescence and immunohistochemistry results showed that the positive expressions of TLR2,TLR4,p-IRAK1,p-STAT3,p-p65 and PD-L1 proteins were increased after heat stress,and some of them were increased in nucleus.Western blotting results showed that proteins in TLR2/4/NF-κB and STAT3 signaling pathway were significantly activated after heat stress(P<0.05),which were consistent with the expressions of animal experiments.WYP significantly reduced the expressions of the above proinflammatory signaling pathways and anti-inflammatory signaling(P<0.05).Conclusion1.The inflammation related pathway of TLR2/4/NF-κB and STAT3 were significantly activated to promote inflammation in the testicular tissues and sertoli cells under heat stress,and the anti-inflammatory signals miR-146a-5p,miR-23a-3p and PD-L1 were also increased in response but played a inhibited role in inflammation.The activation of PD-L1 was regulated by the TLR2/4/NF-κB and STAT3 signaling pathway and played a negative role.2.The miR-146a-5p and miR-23a-3p might play a synergistic inhibitory role in testicular tissues and Sertoli cells under the heat stress,and inhibited the expression of TLR4,p-IRAK1 and NF-κB proteins in TLR2/4/NF-κB and STAT3 signaling pathway and decreased the activation level of inflammatory signaling pathway.3.Wuzi yanzong pill(WYP)might reduce the hyperreactive immune response in heat-stress testis by decreasing the expressions of pro-inflammatory signaling pathway TLR2/4/NF-κB and STAT3 and anti-inflammatory signals miR-146a-5p and miR-23a-3p and PD-L1 in heat-stress testis,thus restored the pro-inflammatory and anti-inflammatory responses to a relatively normal levels.Also,WYP could promote the testis local immune balance,reduce the tissue inflammation damage and provide a favorable immune microenvironment for spermatogenesis. |
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