TLR4 Knockout Alleviates Cadmium Chloride-induced Testicular Injury In Mice And Related Mechanisms

Objective:To explore the effect and mechanism of TLR4 knockout on reducing cadmium chloride-induced testicular injury in mice.Methods:1.Laboratory animals and grouping8-10 weeks old male TLR4 knockout(TLR4 knockout,TLR4^-/-,C57BL/10Sc N)and wild-type mice（wild type,WT,C57BL/10Sc N）were divided into 4 groups:wild-type mice group（WT group）;wild-type mice+cadmium chloride group（WT+Cd Cl₂ group）;TLR4 knockout group(TLR4^-/-group);TLR4 knockout mice+cadmium chloride group(TLR4^-/-+Cd Cl₂ group),8 mice in each group.2.Establish the cadmium chloride exposure modelThe mouse in WT+Cd Cl₂ group and TLR4^-/-+Cd Cl₂ group were given 8 mg/kg of cadmium chloride based on body weight for 15 days.Recording the body weight of the mice.At day 16,the administration was stopped.Then observeing for 3 days.3.Take tissuesOn the 18th day,mice were dissected after sacrifice by CO₂ asphyxiation,take out the liver,spleen,testicles and epididymis of the mouse,weigh the testicles,spleen,liver,and calculate the organ coefficient.The liver,spleen and testicles were fixed on one side of the tissue in 4%paraformaldehyde,and the other part was placed in a cryopreservation tube.Frozen tissues by using liquid nitrogen and then stored them in a-80°C refrigerator4.Sperm quality assessmentObserveing and recording sperm motility,survival rate and malformation rate by using computer-assisted sperm analysis system.5.Hematoxylin-Eosin stainingThe paraffin section of testicular tissue were melted in the oven and then dewaxed and hydrated.After rinsing with distilled water,they were stained with hematoxylene,0.5%hydrochloric acid alcohol,neutralized alkali solution,and then staining cytoplasm by using 0.5%water-soluble eosin.After steeping in gradient alcohol dehydration,xylene transparent,and finally cover slides by Neutral balsam.6.ImmunohistochemistryParaffin sections were melted in an oven at 65°C for 2h.Dewax them in xylene,gradient ethanol,and then gently soaked them in distilled water and PBS.Antigen retrieval by Citrate.Soaked slides by distilled water,PBS,3 times.Use 3%hydrogen peroxide block slides,soaked with distilled water,PBS.Add primary antibody on the tissues,store them at 4°C overnight.The next day,storing them for 2 h at room temperature and after that soak them with PBS,3 times.Add secondary antibody on tissues,37°C,1 h.After DAB,slides were stained in hematoxylin solution for 2 min,and differentiated using 1.5%hydrochloric acid alcohol solution.Gradient ethanol,xylene.Finally,cover slides by Neutral balsam.7.ImmunofluorescenceParaffin sections were melted in an oven at 65°C for 2h.Dewax them in xylene,gradient ethanol,and then gently soaked them in distilled water and PBS.Antigen retrieval by Citrate.Soaked slides by distilled water,PBS,3 times.Use 3%hydrogen peroxide block slides,soaked with distilled water,PBS.Add primary antibody on the tissues,store them at 4°C overnight.The next day,storing them for 2 h at room temperature and after that soak them with PBS,3 times.Add fluorescent secondary antibody dropwise at 37°C,1h,protected from light throughout the process.PBS dip.Cover slides by using anti-fluorescent quenching agent which containing DAPI.8.TUNELAfter dewaxing in xylene at room temperature,dehydration in gradient ethanol,and gentle dip washing in PBS,the experiments were performed by referring to the kit instructions.The slices were covered with anti-fluorescence quenching sealer containing DAPI in a dark environment,and finally photographed and analyzed under confocal laser scanning microscope.9.Real-time Quantitative PCRPrimers were designed and synthesized.Tissue total RNA was extracted and RNA was reverse-transcribed,and the reaction system was set up according to the instructions of the q PCR kit.10.ELISAThe serum of mice was assayed for detecting serum testosterone according to the elisa kit instructions.11.Western Blot Detecting the relative expression levels of TICAM protein in each group of testicular tissue proteins.12.Statistical analysisIBM SPSS 26 processed the data,continuous variable data were represented by mean+S.E.M,and comparisons between groups were performed using an independent sample t-test,multiple comparisons were performed using one-way analysis of variance（ANOVA）.Changes in body weight were perfomed using repeated measurement ANOVA.P<0.05 is significant and P>0.05 is not significant.Results:1.The change of organ index of mice between groups:The analysis of the ratio of weight to body weight of internals organs showed that cadmium chloride led to significant enlargement of spleen in WT+Cd Cl₂group.Compared with the WT control group with a ratio of 0.003±0.0003,the ratio of spleen weight to body weight in the WT+Cd Cl₂ group was 0.0056±0.0008,and the spleen index in the WT+Cd Cl₂group was significantly increased（P<0.001）.The ratio of TLR4^-/-+Cd Cl₂ group was0.0038±0.0013,which was not statistically significant compared with the WT control group.Compared with TLR4^-/-+Cd Cl₂ group,the ratio of spleen weight to body weight of WT+Cd Cl₂ group was significantly increased（P<0.01）.The ratio of liver weight to body weight in the WT+Cd Cl₂ group also changed.Compared with the WT control group,the ratio of the WT+Cd Cl₂group was significantly reduced（P<0.01）.In the testis,there was no significant difference among the four groups.2.Sperm quality assessment found that compared with the WT group,the sperm quality of mice in the WT+Cd Cl₂ group decreased significantly,sperm motility and survival rate decreased significantly,and the sperm malformation rate increased,while the sperm morphology of the TLR4^-/-+Cd Cl₂ group was normal,with no significant changes in sperm viability,survival rate and malformation rate.3.ELISAELISA for serum testosterone found that Cd Cl₂ caused a significant decrease in serum testosterone in mice compared with WT group（P<0.001）and a slight decrease in TLR4^-/-+Cd Cl₂ group,but there was no significant difference.4.Hematoxylin-eosin staining resultsCompared with the WT group,the gap between the spermatogenic tubules enlarged in the WT+Cd Cl₂ group,the diameter of the semigonigenic tubules increased,and vacuoles appeared in the Seminiferous tubule.The Seminiferous tubule in the TLR4^-/-+Cd Cl₂ group were arranged normally,and there was no significant change from the WT group.5.Immunofluorescence and ImmunohistochemicalCompared with the WT group,the expression of S100A8,S100A9,TICAM,IRF3,PTGS2,IL-1βin WT+Cd Cl₂ groups was significantly increased.There was no significant change in the TLR4^-/-+Cd Cl₂ group.6.TUNELThe number of TUNEL positive cells was significantly increased in the WT+Cd Cl₂ group compared with the WT control group,and there was a significant difference（P<0.001）.the number of TUNEL positive cells was significantly increased in the TLR4^-/-+Cd Cl₂ group compared with the WT control group,and there was a significant difference（P<0.01）,but compared with the WT+Cd Cl₂ group,the number of positive cells was less than that of the latter group,and the difference between the two groups was significant（P<0.001）.7.Real-time Quantitative PCRCompared with the WT control group,the expression levels of Caspase-3,Caspase-9,S100A8,S100A9,TRIF,IRF3,NF-κBp65,IL-1β,IL-6 and PTGS2 in the WT+Cd Cl₂ group were increased and the differences were significant,and the expression levels of S100A9,TRIF,and PTGS2 in the TLR4^-/-+Cd Cl₂ group were increased and the differences were significant.8.Western BlotThe results showed that the TICAM protein expression levels were significantly higher in the WT+Cd Cl₂ group and TLR4^-/-+Cd Cl₂ group.Conclusion:1.Cadmium chloride can damage the testes of mice,significantly reduce serum testosterone concentration,and cause a decrease in sperm quality2.TLR4 knockdown can attenuate Cd Cl₂-induced testicular inflammation and germ cell apoptosis in mice,probably by inhibiting the action of S100A8/A9.3.Cadmium chloride activates the TLR4/TRIF signaling pathway in mouse testis.