| OBJECTIVES:(1)To evaluate the intervention effect of kidney-tonifying traditional Chinese medicines on Sertoli cells in animal models with impaired spermatogenesis.(2)To explore the potential molecular mechanism of Qiangjing Tablet in the treatment of male infertility through network pharmacology and molecular docking technology.(3)To observe the protective effect of Qiangjing Tablet on the blood-testis barrier in Sertoli cell of mice with cadmium-induced spermatogenesis impairment,and to explore its mechanism.METHODS:Part Ⅰ: Systematic review of animal experiments to explore the improvement of spermatogenic function by traditional Chinese medicine based on the structure and function of Sertoli cells.Chinese and English databases were searched to screen experimental studies on the structure and function of Sertoli cells in animal models of spermatogenic impairment by traditional Chinese medicine.The risk of methodological bias of the included studies in the literature was assessed using the SYRCLE risk of bias assessment tool for animal experiments,and Meta-analysis of outcome indicators was performed using Rev Man and STATA software.Part Ⅱ: To explore the molecular mechanism of Qiangjing Tablets for the treatment of male infertility based on network pharmacology combined with molecular docking.The active ingredients and potential targets of Qiangjing Tablets were collected using the systemic pharmacology analysis platform of traditional Chinese medicine,and the disease targets of male infertility were screened using Genecard database;then the disease targets were intersected with the predicted drug targets,and the intersection target network and protein-protein interaction network were constructed with the use of Cytoscape software,and the Bioconductor platform and R language were used to GO and KEGG enrichment analysis was performed.The active drug components were also molecularly docked to the disease targets to explore the potential action components and target mechanisms.Part Ⅲ: Experiment 1: 60 male KM rats were randomly divided into 6 groups,blank control group(C group),cadmium chloride model group(MO group),Qiangjing Tablets advance intervention group(QA group),Qiangjing Tablets post-modeling intervention group(QB group),Qiangjing Tablets advance intervention+ post-modeling intervention group(QC group),and positive drug vitamin C advance intervention + post-modeling intervention group(PC group).Before modeling,QA group and QC group were administered with 0.45g/(kg-d)-dose of Qiangjing Tablets by gavage,PC group was administered with 0.2g/(kg-d)-dose of vitamin C by gavage,NC group,MO group and QB group were administered with saline by gavage for 10 d.2.0mg/(kg-d)-dose of cadmium chloride was used for intraperitoneal injection of modeling.After moulding,QB and QC groups were given 0.45g/(kg-d)Qiangjing Tablets by gavage,PC group was given 0.2g/(kg dose of vitamin C by gavage,NC and MO groups were given the same amount of saline by gavage for 10 d.Body weight,wet weight of testes and epididymis were recorded,and sperm count,sperm motility and sperm survival rate were measured;structural changes of testes were observed by HE staining and electron microscopy.The ultrastructure of the testis and changes in the blood-testis barrier were observed by electron microscopy.The anti-sperm antibodies in mice serum and testis tissues were measured,and the changes of blood-testis barrier permeability were observed by fluorescence tracing method.Experiment 2: Sixty male KM mice were divided into 6 groups of 10 mice each according to the random number table method,namely,control group(C group),model group(MO group),Qiangjing Tablets group(QJP group),positive drug group(VC group),Qiangjing Tablets + inhibitor group(QJP + Y group),and inhibitor group(Y group).Intraperitoneal injection of cadmium chloride at a dose of 2.0 mg/kg was used for modeling.After modeling,QJP group and QJP+Y group were given 0.45g/(kg-d)of strong semen tablets by gavage intervention,VC group was given 0.2g/(kg-d)of vitamin C by gavage intervention,C group and MO group were given the same amount of saline by gavage,and QJP+Y group and Y group were given 4mg/(kg-d)dose of JR-AB2-011 by tail vein injection(m TORC2-specific inhibitor)was administered for 10 d.The expression and localization of blood-testis barrier-related proteins N-cadherin,Occludin,ZO-1,Claudin-11,F-actin,andβ-Tubulin in testicular tissue were determined by immunofluorescence,and Cdh2,Ocln,Tjp1,Cldn11,Nexn,Tubb3 m RNA conten were determined by q PCR.Immunohistochemistry was used to determine the expression of HGF,PI3 K,AKT,Rictor and Raptor,the key targets of signaling pathway,and q PCR was used to determine HGF,PI3 K,AKT,Rictor and Raptor m RNA content.RESULTS:Part Ⅰ: A total of 30 studies were included.meta-analysis results showed that herbal medicine increased sperm density and increased sperm motility compared with the model group.Increased Sertoli cells cytoskeleton-related testicular tissue Vimentin levels,Vimentin m RNA levels.Increase blood-testis barrier tight junction-related testicular tissue Occludin,ZO-1,Claudin-11 level;increase blood-testis barrier adherent junction-related testicular tissue β-catenin,N-cadherin level;increase blood-testis barrier gap junction-related testicular tissue Cx43 level.Improving Sertoli cells function,increasing serum INHB level,increasing Sertoli cells markers FSHR,FSHR m RNA,INHB m RNA,ABP m RNA,TF level in testicular tissue,increasing support cell homing function related SCF,SCF m RNA,GDNF,GDNF m RNA,BMP4,BMP4 m RNA level in testicular tissue.Part Ⅱ: A total of 109 active ingredients and 286 targets for subsequent analysis were obtained for strong sperm tablets,476 targets for male infertility,and 69 common drug-disease targets were mapped.KEGG pathway analysis suggests that it is mainly related to PI3K-Akt,TNF,endocrine resistance and other signaling pathways,and PI3K-Akt is the core signaling pathway.The active ingredients quercetin,lignan,kaempferol,β-glutamyl sterols,and arachidonic acid in the strong essence tablets bind stably to the target proteins MAPK1,AKT1,HSP90AA1,IL6,and ESR1.Part Ⅲ: Experiment 1: on days 14 and 21(i.e.,post-modeling),mice in MO,QA,QB,QC,and PC groups showed a decrease in body weight compared to group C(P <0.05).Testicular index,compared with group C,decreased in all groups(P < 0.05),compared with group MO,testicular index increased in groups C,QA,QB,QC and PC with statistically significant differences(P < 0.05).Epididymal index,compared with group C,epididymal index decreased in group MO,group QA,group QB and group PC(P < 0.05).Compared with group MO,epididymal index increased in group QA,group QB,group QC and group PC,and the difference was statistically significant(P < 0.05).Sperm quality: compared with group C,sperm density,sperm motility and sperm survival rate appeared to be decreased in each group(P < 0.05);compared with group MO,sperm density,sperm motility and sperm survival rate increased in groups C,QA,QB,QC and PC(P < 0.05).Testicular HE staining showed large testicular necrosis in the MO group,and localized testicular tissue necrosis of different degrees in the QA,QB,QC,and PC groups.Johnsen score of testicular tissue was decreased in other groups compared with C group(P < 0.05),and Johnsen score was increased in C group,QA group,QB group,QC group and PC group compared with MO group(P < 0.05).Electron microscopy showed that the MO group showed severe damage to the germinal tubules,mesenchymal cells and blood-testis barrier.the QA,QB,QC and PC groups showed different degrees of structural damage to the germinal tubules,mesenchymal cells and blood-testis barrier.The serum Serum antisperm antibody level were increased in all groups compared with group C(P < 0.05),and decreased in group C,QA,QB,QC and PC compared with group MO(P < 0.05).Asab levels in testis tissues were increased in MO,QA and QB groups compared with group C(P < 0.05),and decreased in group C,QA,QB,QC and PC groups compared with group MO(P < 0.05).The green fluorescence in group MO partially penetrated through the basement membrane to the inside of the germinal tubules,the blood-testis barrier structure was destroyed and the permeability of the blood-testis barrier was greatly increased in this group.Experiment 2: Immunofluorescence detection showed that N-cadherin was distributed around spermatogenic cells and Sertoli cells in testis tissues of mice in the C group;Occludin was located between Sertoli cells.ZO-1 is linearly distributed near the basement membrane.Claudin-11 is located around spermatogenic cells.F-actin is located around the spermatogenic cells and the basement membrane.β-Tubulin is located around spermatogenic cells and testicular supporting cells.Compared with the MO group,the QJP group restored the localization of N-cadherin,ZO-1,Claudin-11,and F-actin at the base of the seminiferous tubules,and Occludin and β-Tubulin in the Sertoli cells of the testis.Compared with the C,VC,and QJP groups,the expression of N-cadherin,ZO-1,Occludin,Claudin-11,F-actin,and β-Tubulin was decreased in the MO,QJP+Y,and Y groups.q PCR assays showed that compared with the C group,Cdh2,Ocln,Tjp1,Cldn11,Nexn,Tubb3 m RNA expression were decreased in all groups(P < 0.05).Cdh2,Ocln,Tjp1,Cldn11,Nexn,Tubb3 m RNA expression increased in the VC and QJP groups compared with the MO,QJP+Y,and Y groups(P< 0.05).Immunohistochemistry showed that the percentage of positive immunohistochemical area for HGF,PI3 K,AKT,and Rictor was decreased(P < 0.05)and the percentage of positive immunohistochemical area for Raptor was increased(P< 0.05)in all groups compared with group C.Compared with MO group and Y group,the percentage of positive immunohistochemistry area for HGF,PI3 K and AKT was increased in VC group,QJP group and QJP+Y group(P < 0.05),and the percentage of positive immunohistochemistry area for Raptor was decreased(P < 0.05).The percentage of Rictor immunohistochemical positive area was increased in VC group and QJP group compared with MO group,Y group and QJP+Y group(P < 0.05).q PCR assay showed that the content of HGF,Pik3 cb and Akt1 m RNA was decreased in MO group and Y group compared with C group(P < 0.05),and the content of HGF,Pik3 cb and Akt1 m RNA was decreased in VC group,QJP group and QJP+ group compared with C group,MO group and Y group.HGF,Pik3 cb,and Akt1 m RNA contents were increased in the VC,QJP,and QJP+ groups compared with the C,MO,and Y groups(P < 0.05).Rictor m RNA content was decreased in all groups compared with group C(P < 0.05),and Rictor m RNA content was increased in groups C,VC,and QJP compared with groups MO,Y,and QJP+ Y(P < 0.05).Raptor m RNA content was increased in MO group and Y group compared with C group(P < 0.05),and Raptor m RNA content was decreased in VC group,QJP group and QJP+Y group compared with C group,MO group,Y group and QJP+Y group(P < 0.05).CONCLUSION: Systematic review studies have shown that kidney-tonifying traditional Chinese medicines can effectively improve sperm density and motility in animal models with impaired spermatogenesis,and its mechanism of action is related to improving the structure and function-related targets of Sertoli cells.Qiangjing Tablet,representative of invigorating the kidney and activating blood can treat male infertility through multi-network and multi-target.Its active ingredients have high stable binding to key target proteins of the disease,and PI3K-Akt is one of its core pathways.Through experimental verification,it was found that the advanced intervention,post-modeling intervention and whole-process intervention of Qiangjing Tablet all have a good protective effect on the reproductive organs and function of the cadmium chloride-induced mouse testicular injury model,which reflects the traditional Chinese medicine Taking Precaution Against Disease,And Preventing The Development Of The Occured Disease.Protecting the blood-testis barrier is one of the mechanisms of Qiangjing Tablet’s anti-Cd Cl testicular injury.The protective effect of Qiangjing Tablets on the blood-testis barrier may be related to the up-regulation of HGF,PI3 K and AKT expression on RTK/PI3K/Akt/m TOR pathway and the restoration of Rictor/Raptor balance. |
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