The Roles Of The P2RX7-Mediated Inflammation And Apoptosis In Uterine Tissue Damage In Mice Induced By Cigarette Smoke Exposure

Objective: Cigarette smoke(CS)contains thousands of harmful substances such as nicotine,tar,CO,which can damage multiple systems of the human body.In recent years,the number of women who smoke actively and passively has increased worldwide,and the smoking rate of women of childbearing age has shown a significant upward trend.However,many women of childbearing age have not realized the damage caused by smoking to female fertility.Studies have found that smoking can lead to impaired uterine development in women,such as affecting the process of decidualization of the endometrium,making the endometrium irregularly mature,and reducing the implantation rate of fertilized eggs.This study mainly explored the mechanism of CS exposure resulting in uterine damage and the clinical targets that can treat women with CS exposure.Histone deacetylase(HDAC)has received wide attention as a therapeutic target for many diseases.The previous results of this research group also showed that CS exposure can cause histomorphologic alterations in uterus by activating HDAC1/2,while HDAC inhibitor trichostatin A(TSA)can reactivate autophagy to alleviate CS exposure-induced uterine histomorphology alterations.CS contains a variety of harmful substances,so whether TSA can reduce uterine damage caused by CS exposure through other routes requires further exploration.P2RX7 is mainly found in immune cells,and its overexpression is involved in many physiological/pathophysiological processes.P2RX7 can mediate inflammation and apoptosis,and is P2RX7 receptor-mediated inflammation and apoptosis involved in uterine damage induced by CS exposure? Does the P2RX7-mediated inflammation and apoptosis participate in the relief of uterine tissue damage caused by CS exposure by TSA? Does A438079(P2RX7 inhibitor)alleviate uterine tissue damage induced by CS exposure in mice? At present,further exploration is needed.Methods:(1)Experimental grouping and specimen collection: 60 healthy C57BL/6female mice of SPF grade were divided into 5 groups,namely control,CS,CS+TSA,CS+Bz ATP(P2RX7 agonist)and CS+A438079(P2RX7 inhibitor).The weight changes of mice in each group were recorded during CS exposure.After 30 days of CS exposure,the uterine tissues of at least 5 C57BL/6 female mice were taken from each group,and the uterine tissues at the estrous stage were weighed,fixed or-80°C cryopreserved.(2)Alterations in uterine tissue structure of mice after CS exposure: HE staining was used to observe alterations in uterine tissue structure of mice after CS exposure.(3)Expression of inflammation and apoptosis-related proteins in uterine tissue of mice after CS exposure: Western blot was used to detect P2RX7,NLRP3 inflammatory factors(ASC,NLRP3,IL-1? and cleaved caspase1)and apoptosis-related proteins(BCL-2,cleaved caspase3 and cleaved caspase9)in mice uterine tissue after CS exposure.(4)Expression of inflammation and apoptosis-related proteins in uterine tissue of mice after TSA treatment: Western blot was used to detect P2RX7,NLRP3 inflammatory factors and apoptosis-related proteins in uterine tissue of CS exposed mice after TSA treatment.(5)m RNA levels of NLRP3 inflammatory factors after TSA treatment: Real time-PCR method was used to detect the m RNA expression levels of Hdac1,P2rx7,Asc and Il-1? in uterine tissue of CS exposed mice after TSA treatment.(6)Structural changes of uterine tissues of CS exposed mice after A438079 treatment:The HE staining method was used to observe the structural changes of uterine tissues of CS exposed mice after A438079 treatment.(7)Expression of inflammation proteins in CS exposed mice after A438079 treatment: Western blot was used to detect the expression of P2RX7 and NLRP3 inflammatory factors in mice uterus;immunohistochemistry was used to detect the expression of ASC and IL-1? in CS exposed mice uterus;immunofluorescence double staining was used to observe the infiltration of inflammatory cells in CS exposed mice uterus.(8)Expression of apoptosis-related proteins in uterine tissue of CS exposed mice after A438079treatment: Western blot was used to detect the expression levels of apoptosis-related proteins in uterine tissue of CS exposed mice after A438079 treatment.Results:(1)CS exposure resulted in histomorphologic alterations of mice uterus:After CS exposure,the muscle layer of mice uterine tissue became significantly thinner,and the number of interstitial cells and glands decreased significantly.(2)CS exposure caused inflammation and apoptosis in mice uterine tissue: CS exposure induced P2RX7,NLRP3 inflammatory factors(NLRP3,ASC,IL-1? and cleaved caspase1)and apoptosis-related proteins(BCL-2,cleaved caspase3 and cleaved caspase9)were significantly up-regulated in mice uterine tissue.(3)TSA alleviates the inflammation and apoptosis of mice uterine tissues induced by CS exposure: TSA inhibited the expression up-regulated of P2RX7,NLRP3 inflammatory factors and apoptosis-related proteins in mice uterine tissue induced by CS exposure.(4)TSA alleviates the m RNA transcription activity of NLRP3 inflammatory factors in mice uterine tissue induced by CS exposure: TSA significantly inhibited the increase of m RNA transcription levels of Hdac1,P2rx7,Asc and Il-1? in mice uterus induced by CS exposure.(5)A438079improved histomorphologic alterations of mice uterine induced by CS exposure:A438079 alleviates mice's weight loss and reduced uterine tissue wet weight caused by CS exposure.HE staining results showed that A438079 significantly alleviated the thinning of the myometrium and reduced the number of interstitial cells and glands in mice caused by cigarette smoke exposure.(6)A438079 inhibited CS exposure induced uterine tissue inflammation: Western blot results showed that A438079 significantly inhibited the up-regulation of the expression of P2RX7 and NLRP3 inflammatory factors in uterine tissue of mice induced by CS exposure;Immunohistochemical results showed that A438079 inhibited activation of inflammatory cells IL-1? and ASC induced by CS exposure;Immunofluorescence double staining showed that A438079 inhibited the increase of inflammatory cell infiltration in uterine tissue induced by CS exposure.(7)A438079 inhibited CS exposure induced apoptosis in mice uterine tissue: A438079 significantly inhibited the expression of apoptosis-related proteins(BCL-2,cleaved caspase3 and cleaved caspase9)in uterine tissue induced by CS exposure.Conclusion: 1.CS exposure results in histomorphologic alterations in uterine tissues of mice.2.P2RX7-mediated inflammation and apoptosis participate in the uterine damage process of mice induced by TSA relieved cigarette smoke exposure.3.A438079 improves the histomorphologic alterations of uterine tissue caused by CS exposure by inhibiting P2RX7-mediated inflammation and apoptosis.