The Mechanism Of PP1A Inhibiting Pyroptosis Of Mice Adipocytes By Reducing ROS Level

As a new type of programmed cell death?PCD?,pyroptosis has attracted much attention.Studies have shown that a variety of exogenous stimuli can cause pyroptosis.Pyroptosis is considered to be of great significance in the development of cancer.Caspase in cancer cells can respond to chemotherapy drugs,and then cut the Gasdermin family proteins to expose the N-terminus.The N-terminal sequence and cell membrane are bound to form a pore-like structure on the surface of the cell membrane,which changes the internal osmotic pressure of the cell,causing leakage of cell contents and inflammatory death of cell.Studies have shown that pyroptosis also occurs in adipocytes.Serine/threonine protein phosphatase 1catalytic subunit alpha?PP1A?is a dephosphatase located in the nucleus and plays an important role in cell development regulation,glucose metabolism,and protein synthesis.PP1A and its family of proteins PP2A and PP4 were found to be able to use their phosphatase activity to participate in regulating DNA damage checkpoints and repair of DNA damage.PP1A can also target important proteins in the process of apoptosis to participate in the regulation of apoptosis.However,it is unclear whether pyroptosis,an important physiological process of adipose,is regulated by PP1A.This study explored the role and mechanism of PP1A in inhibiting adipocytes pyroptosis by alleviating ROS of adipocytes by proteomics sequencing,q PCR,western blotting,immunofluorescence,immunohistochemistry and other methods.This test provides a certain theoretical basis for analyzing the regulatory function and mechanism of PP1A in cell scorch caused by oxidative stress in adipocytes.The results of this study are as follows:1. PP1A inhibits adipocytes pyroptosis by relieving ROS level.By Leptin-treated adipocytes proteome sequencing,PP1A expression level was screened with significantly reduced under the control of Leptin?P<0.01?,and the effect of PP1A on adipocytes pyroptosis was studied.H₂O₂ was used to construct a ROS model of adipocytes,and the expression levels of ROS and the activities of SOD,MDA,and CAT in adipocytes were tested to verify that the ROS model was successfully constructed?P<0.05?.Co-localization staining of ROS and Mitotracker in adipocytes revealed that ROS levels were significantly reduced under PP1A overexpression treatment?P<0.05?.JC-1 staining and detection of mitochondrial respiratory chain function genes m RNA levels showed that PP1A can alleviate mitochondrial dysfunction caused by ROS?P<0.05?.Under the ROS model,PP1A can down-regulate the m RNA levels of the pro-apoptotic genes Bad,Bax,Caspase9 and Caspase3,increase the m RNA levels of the anti-apoptotic gene Bcl-2,inhibit the expression of pyroptosis gene GSDME,and inhibit the activation of cleaved-Caspase3.Decreased the protein level of GSDME-N?P<0.05?.PP1A can effectively inhibit the transfer of cytochrome c from mitochondria to cytoplasm?P<0.01?.Tunnel staining showed that overexpression of PP1A could inhibit DNA damage in adipocytes?P<0.05?;reduce LDH content in cell culture fluid,and reduce leakage of IL-1?in cells?P<0.05?,shows that PP1A can inhibit the pyroptosis of adipocytes.2. PP1A prevents adipocytes pyroptosis by inhibiting the ROS/MAPK/Caspase3pathway.H₂O₂ was used to construct a ROS model of adipocytes,and the intracellular ROS content was detected under the model.The ROS level in the cells after NAC treatment was significantly reduced?P<0.05?.Under the ROS model,the phosphorylation levels of MAPK pathway proteins were significantly increased,and they were significantly decreased after NAC treatment?P<0.05?.Further exploring the protein expression downstream of MAPK,it was found that under the ROS model,the protein level of cleaved-Caspasae3 was significantly increased,and the protein level of GSDME-N was also significantly increased?P<0.05?.After treatment with three inhibitors of the MAPK pathway,The protein levels of cleaved-Caspasae3 and GSDME-N were reduced to varying degrees?P<0.05?;and under the ROS model,the levels of IL-1?and LDH in adipocyte culture fluid were significantly increased,but both were reduced with MAPK pathway inhibitors treatments.There are different degrees of reduction?P<0.05?.When over-expressing PP1A treated adipocytes,the protein expression levels of p-ERK,p-JNK,p-p38,cleaved-Caspase3 and GSDME-N were significantly reduced,but the opposite was observed with si PP1A treatment?P<0.05?.The expression levels of cleaved-Caspase3 and GSDME-N by immunofluorescence were consistent with the above results?P<0.05?.The above results indicate that PP1A prevents adipocytes pyroptosis by inhibiting the ROS/MAPK/Caspase3 pathway.3. PP1A relieves adipocytes pyroptosis through inhibiting adipose tissue M1macrophage polarization.H₂O₂ treatment significantly promoted the m RNA levels of M1macrophage markers TNF?and IL6 in adipose tissue,while inhibiting the expression of M2macrophage markers CD206 and IL10.Overexpression of PP1A just reversed this phenomenon and inhibited M1 macrophage polarization?P<0.05?.The protein expression levels were consistent with the m RNA levels.Adipose tissue HE staining showed that PP1A can effectively reduce the size of adipocytes,and promote the m RNA expression levels of lipolytic genes ATGL,HSL and CPT-1 in adipose tissue,and inhibit the expression levels of adipose synthesis genes ACC and FASN?P<0.05?.H₂O₂ treatment promoted M1 macrophage marker gene expression,si PP1A treatment further promoted M1 macrophage polarization,and treatment with ROS-specific inhibitors could effectively inhibit M1 macrophage polarization?P<0.05?.After polarization of M1 macrophages,m RNA levels of ROS-related genes CAT,SOD-2 and pro-apoptosis-related genes Bad,Bax,Caspase3 and pyroptosis-related genes GSDME,GSDMD were significantly up-regulated in adipose cells,and the apoptosis suppressor gene Bcl-2 m RNA levels were significantly down-regulated,and the protein expression levels of Bad,Bax,cleaved-Caspase3,and GSDME-N were also significantly increased,and these were reversed after treatment with overexpression of PP1A,the immunohistochemistry of GSDME-N is also consistent with it.The results of this experiment show that PP1A can inhibit the pyroptosis of adipocytes by promoting the polarization of M2 macrophages in adipose tissue.This study demonstrated that PP1A relieves fat cell scorch death by inhibiting the ROS/MAPK/Caspase3 pathway.PP1A inhibits adipocytes pyroptosis by inhibiting M1macrophage polarization in adipose tissue,regulating the balance of M1/M2 macrophages and the interaction of adipocyte and macrophages.