NF-?B Signaling Pathway Mediates Autophagy To Participate In The Mechanism Of Male Reproductive Damage Induced By PM2.5

BackgroundFine particulate matter PM2.5 refers to solid or liquid particulate matter with an aerodynamic diameter of 2.5 ?m or less suspended in the air.PM2.5 can cause damage to human respiratory system,cardiovascular system,immune system and nervous system,causing the occurrence of related diseases.Studies have shown that PM2.5 exposure is closely related to reproductive dysfunction,but the mechanism of damage has not been clarified.NF-?B is a widely expressed inflammatory regulatory transcription factor.Autophagy regulates many important physiological processes.NF-?B and autophagy,as important factors involved in the regulation of inflammatory response,are closely related.However,whether NF-?B and autophagy are involved in PM2.5 induced reproductive damage has not been reported.Therefore,the mechanism of NF-?B and autophagy in the reproductive injury caused by PM2.5was discussed in this paper.PurposeThis study intends to explore the role of NF-?B signaling-mediated cell autophagy in PM2.5-induced reproductive damage through a combination of in vivo animal experiments and in vitro cell experiments.Methods1.Large flow sampler was used to collect PM2.5 and quartz fiber filter membrane sampling.The mass concentration was analyzed and the contents of 9metal elements were determined by inductively coupled plasma mass spectrometry(ICP-MS).2.Selection and grouping of experimental animals60 healthy four-week-old male Sprague-Dawley(SD)rats were divided into control group,PDTC group,PM2.5 group,PM2.5+PDTC group,the time of exposure was 4 weeks.3.Determination of organ weight and injury-related indicators caused by PM2.5The weight of testicular and epididymal tissues was separated and the viscera coefficient and sperm quality parameters were calculated.Serum follicle stimulating hormone(FSH),luteinizing hormone(LH)and testosterone(T)were detected by ELISA.Histopathological changes of testis were observed by HE staining.Cell apoptosis in testicular tissues was detected by TUNEL and fluorescence quantitative PCR(real-time PCR)method.Biochemical analysis was used to detect oxidative stress related indexes in testicular tissues.4.Detection of heavy metal elements in rat visceraICP-MS was used to determine the metallic elements in lung,blood,heart and testicular tissues of each group rats.5.Primary cells were isolated and cultured to determine the toxic concentration of PM2.5Three weeks old male SD rats were isolated,cultured,purified and identified testicular support cells.CCK-8 was used to detect cell activity after exposure to PM2.5 at different doses,and determined the subsequent exposure dose and time in combination with IC50.6.Molecular detection of related effects of NF-?B signaling pathwayThe groups were divided into control group,PDTC group,PM2.5 group and PM2.5+PDTC group.The levels of NF-?B and inflammatory factors were detected by RT-PCR and Western blot.7.Cell proliferation,apoptosis and cycle level changes were detected after treatment with autophagy inducer Rapamycin and autophagy inhibitor 3-MA.8.Detection of autophagy response statusAutophagosome was detected by transmission electron microscopy.RT-PCR and Western blot were used to detect the expression of autophagy related markers.Results1.PM2.5 concentration in each month exceeded the limit of ambient air quality,and Cr,and As elements exceed their air environment target values.2.Compared with the control group,the weight(F=1.75,P=0.045),testicular weight(F=3.96,P=0.038)and epididymis(F=4.52,P=0.018)of the rats in the PM2.5group were all reduced.The sperm density(F=10.66,P=0.022)and survival rate(F=14.98,P=0.012)of the rats were significantly decreased,while the deformity rate(F=21.25,P=0.006)was significantly increased.3.Microscopic examination of testicular tissues with HE staining showed that the testicular structures of the control group and PDTC group were intact.In the PM2.5 group,the spermatogenic epithelial cells were loosely intercellular,and there were exfoliated spermatogenic epithelial cells in the lumen.After combined action with PDTC,the exfoliated cells in lumen decreased significantly.4.The results showed that compared with the control group,the apoptosis rate of PM2.5 group was significantly increased,and the difference was statistically significant(t=5.75,P=0.029).Compared with that in the PDTC combined group,the apoptosis level was decreased,and the difference was statistically significant(t=4.88,P=0.040).5.Serum follicle stimulating hormone: compared with the control group,the content of serum follicle stimulating hormone in PDTC group(t=6.23,P<0.0001),PM2.5 group(t=6.72,P<0.001)and PM2.5+PDTC group was decreased(t=8.02,P<0.001).Luteinizing hormone: compared with the control group,there was no significant change in serum luteinizing hormone content in PDTC group,PM2.5group and PM2.5+PDTC group.Testosterone: compared with the control group,the testosterone content of PM2.5 group decreased(t=6.86,P<0.001).Compared with the PM2.5 group,the testosterone content of the PDTC combined group increased(t=4.39,P<0.001).6.The expression of oxidative stress in testicular tissue: PM2.5 exposure promoted the expression level of MDA and reduced the activity of SOD and GSH-PX.The expression level of MDA was decreased by PDTC(F=24.14,P=0.000),while the activity of SOD(F=47.28,P=0.000)and GSH-PX(F=15.13,P=0.001)was increased.The expression of oxidative stress in testicular cells showed that the levels of MDA and SOD in 25?g/ml and 50?g/ml PM2.5 groups were not significantly different from those in the control group,while the levels of MDA and SOD in 100?g/ml,200?g/ml and 400?g/ml PM2.5 groups were significantly increased after treatment.7.The expression levels of NF-?B and inflammation-related marker m RNA and proteins in testis and testicular Sertoli cells showed increased levels of NF-?B signaling and downstream inflammation-related effector molecules.PDTC effectively antagonized the activation of NF-?B signaling pathway and reduced inflammatory response,with statistically significant differences(P<0.05).8.Expression of autophagy-related marker m RNA and proteins in testicular tissue and testicular Sertoli cells.The results showed that PM2.5 treatment significantly increased the expression levels of Beclinl,P62 and LC3,while PDTC effectively antagonized the autophagy reaction of cells,and the difference was statistically significant(P<0.05).The autophagosome was detected by transmission electron microscopy and the same results were obtained.9.By detecting the contents of metal elements in the lungs,blood,heart and testicles of the rats,it was found that compared with the control group,the levels of 7metals in lung tissues,6 metal elements in blood,4 elements in testes and heart were increased in the PM2.5 group.When PDTC was added to PM2.5 exposure,the contents of Mg,Cu,Cd and Pb in lung decreased.The contents of Mg,Mn,Cd and Pb in blood decreased.The contents of Cr,As and Cd in heart decreased.The contents of Cr,As and Cd in testis decreased.10.The survival rate and cell proliferation of testicular sertoli cells was inhibited in the PM2.5 treatment group,with statistically significant differences(t=4.25,P<0.05).11.The autophagy inducer rapamycin aggravated the effect of PM2.5 on inhibiting cell proliferation(t=3.54,P=0.005)and promoted apoptosis(t=4.54,P=0.045),while the autophagy inhibitor slowed down the effect of PM2.5 on inhibiting cell proliferation(t=2.80,P=0.019)and inhibited apoptosis(t=10.59,P=0.009).PM2.5 exposure could cause S-phase arrest,and S-phase arrest was more pronounced when rapamycin was added(t=4.21,P=0.042);on the contrary,the percentage of S-phase cells in PM2.5 group was lower when 3-MA was added(t=3.84,P=0.045).Conclusion1.From both in vivo and in vitro experiments,it has been confirmed that PM2.5has significant damage effect on male reproductive function.2.PM2.5 exposure will induce oxidative stress,activate the NF-?B signaling pathway,and further cause inflammation,and at the same time activate the autophagy program.3.The autophagy response mediated by NF-?B signaling pathway further activates cell apoptosis,inhibits cell proliferation and causes cell cycle arrest,which ultimately leads to tissue cell damage.4.The heavy metal elements Cr,As,Cd in PM2.5 may be closely related to the damage of male reproductive function.