Mechanism By Which Exosomes Derived From Diabetic Nephropathy Podocyte Promote Mesangial Cells Injury And The Effect Of Berberine

Diabetic nephropathy(DN)is considered to be one of the most common and serious complications of diabetes mellitus(DM),and also one of the main causes of end-stage renaldisease(ESRD).Mesangial cells(MCs)and podocytes are two types of cells inherent in the glomerulus,and their abnormal functional changes play a key role in the development of DN.Exosomes are extracellular vesicles containing proteins,DNA,RNA,inflammatory factors and other substances,and almost all cells secrete exosomes.Previous studies have shown that MCs induced by high glucose(HG)may affect the function of podocytes through the release of exosomes,and the supernatant of podocytes stimulated by HG can cause damage to the function of MCs,suggesting that podocytes may affect the function of MCs through exosomes.TLR4/NF-κB/NLRP3 plays a key role in the abnormal proliferation of MCs.Nuclear factor-kappa B(NF-κB)is a transcription factor that regulates a series of genes.When stimulated,it will translocate from the cytoplasm to the nucleus into the nuclear NF-κB and gene promoters Binding of the regulatory region and participate in the expression of inflammatory genes.Berberine(BBR)is an isoquinoline alkaloid,which is an effective ingredient extracted from the traditional Chinese medicine Coptis.It has various pharmacological effects such as antioxidant activity,regulating blood lipids and reducing inflammation.The above studies indicate that podocytes stimulated by HG may affect the function of MCs through exosomes,and its effect may be achieved by regulating the TLR4/NF-κB/NLRP3 signaling pathway.Therefore,this project intends to extract exosomes released by podocytes by ultracentrifugation,and after co-cultivation with MCs in vitro,observe the functional changes of MCs and the expression levels of TLR4/NF-κB/NLRP3 signaling pathway-related proteins to study the HG stimulation The mechanism of podocyte-derived exosomes causing MCs damage,and the important role of BBR in improving the function of MCs,to further explore the role of exosomes in the signal transduction between MCs and podocytes,which is BBR The treatment of DN provides a modern scientific basis and opens up new ideas for providing new targets for drugs.Objective1.Determine whether the podocyte-derived exosomes stimulated by HG have a damaging effect on MCs.2.Determine whether BBR can improve MCs damage caused by podocyte-derived exosomes stimulated by HG.3.Determine whether the mechanism by which BBR protects HG-induced podocytederived exosomes from damage to MCs is related to the regulation of the TLR4/NF-κB/NLRP3 pathway.MethodsAfter collecting a sufficient amount of podocyte supernatant,use ultracentrifugation to extract appropriate amount of exosomes,use Western blot to observe the expression of related marker proteins of exosomes;use transmission electron microscope to observe the size and morphological characteristics of exosomes;A laser confocal microscope was used to observe the uptake of exosomes by MCs.The podocytes and MCs were co-cultured and divided into normal group(NC),HG model group and HG+GW4869(exosome inhibitor)group.After 24 hours,the proliferation of MCs was observed by high-content cell imaging method,and fluorescence was used.Microscopic observation of reactive oxygen species(ROS)levels and Western blot method to detect the expression of related deposited proteins,laser confocal detection of P65 nucleus level,to observe whether HG-induced podocyte-derived exosomes participate in the damage of MCs function.The CCK-8 method was used to determine the optimal concentration and time for the influence of exosomes to stimulate MCs.CCK-8 method and high-content cell imaging method were used to detect the effect of BBR high and low dose groups on the proliferation of MCs.Western blot and laser confocal were used to detect the effect of BBR high and low dose groups on the expression of related proteins on MCs.Western blot and laser confocal methods were used to detect whether TLR4,NF-κB and NLRP3 inflammasome pathways could form signal transduction axes.CCK-8 method,high-content cell imaging method,laser confocal method and ROS were used to detect whether BBR could improve the function of MCs by adjusting the signal axis.Results1.The precipitates obtained by differential and hypervelocity centrifugation were podocyte-derived exosomesWestern blot results showed that the extracted material had the expression of exosomal marker molecules CD63 and TSG101,but the endoplasmic reticulum molecular chaperone protein Calnexin hardly expressed.Transmission electron microscopy results show that the appearance of the proposed material is a double-layer film-like structure,and the size is about 50～150 nm,which is consistent with that reported in the literature.2.Podocyte-derived exosomes were absorbed by MCsThe results of laser confocal microscopy showed that MCs could effectively phagocytosis PKH67 labeled exosomes.3.HG induces podocyte-derived exosomes to participate in the impairment of MCsfunctionThe results of cell co-culture showed that compared with the normal group,the abnormal proliferation of MCs,ROS,related deposition protein and p65 levels in the HG group were significantly increased;Compared with the HG group,GW4869(exosome inhibitor)can inhibit the abnormal proliferation of MCs,ROS,related deposition protein and p65 levels,indicating that podocyte-derived exosomes may be involved in the damage of MCs function.4.Optimal conditions for the effect of podocyte-derived exosomes on MCsThe results of CCK-8 method showed that compared with the normal exosomes group,60 μg/m L exosomes stimulated MCs for 24 h,and the proliferation of MCs was more significant,which can be used as the best condition for stimulating MCs.5.BBR significantly improved MCs damage induced by exosomes of podocyte derived from high glucoseCCK-8 method and high-content cell imaging detection of cell proliferation showed that compared with the normal exosomes group,the proliferation level of MCs in the high-glycemic exosomes group was significantly increased,and compared with the HG exosomes group,the BBR level was low The dose group could effectively reduce the proliferation of MCs to varying degrees;The results of Western blot and laser confocal detection showed that compared with the normal exosomes group,the expression of the upper-associated deposition protein of MCs in the high-glycemic exosomes group was significantly up-regulated;compared with the HG exosomes group,the BBR high-and low-dose group.6.BBR may improve the damage of podocyte-derived exosomes to MCs induced by HG,which may be related to TLR4/NF-κB/NLRP3 signaling pathwayWestern blot results showed that compared with the normal exosomes group,the protein expression levels of TLR4,p-IκB,p-p65 and NLRP3 in the HG exosomes group were significantly increased.Compared with the HG exosomes group,BBR high and low dose groups could effectively down-regulate the expression levels of TLR4,p-IκB,p-p65 and NLRP3 protein to varying degrees.7.TLR4,NF-κB and NLRP3 inflammasome pathways form signal transduction axis,and BBR modulates this signal axis to improve MCs injuryWestern blot results showed that,compared with the normal exosomes group,the expression of TLR4,p-p65 and NLRP3 protein in the HG exosomes group was increased;compared with the HG exosomes group,BBR and TAK-242 could improve Effectively down-regulate the protein expression levels of TLR4,p-p65 and NLRP3.The laser confocal results showed that compared with the normal exosomes group,the nuclear translocation of p65 in MCs in the HG exosomes group was significantly increased;compared with the HG exosomes group,BBR and TAK-242 could effectively inhibit p65 in MCs nuclear translocation.After MCs plus podocyte-derived exosomes were incubated with BBR,TAK-242,PDTC and TAK-242+PDTC,Western blot results showed that compared with the normal exosomes group,the HG exosomes group the expression of NLRP3 protein was significantly increased.Compared with the HG exosomes group,BBR,TAK-242,PDTC and TAK-242+PDTC could effectively down-regulate the expression of NLRP3 protein to varying degrees.8.BBR regulates the TLR4/NF-κB/NLRP3 inflammasome signaling pathway and inhibits the injury of podocyte-derived exosomes to MCsThe results of CCK-8 method and high-content cell imaging method showed that compared with the normal exosome group,the proliferation level of MCs in the HG exosome group was significantly higher;compared with the HG exosome group,BBR,TAK-242,PDTC and TAK-242+PDTC can effectively reduce the proliferation of MCs to varying degrees,and inhibit the damage of podocyte-derived exosomes to MCs.The results of laser confocal detection showed that compared with the normal exosomes group,the expression of MCs related deposition proteins in the HG exosomes group was significantly increased;compared with the HG exosomes group,BBR,TAK-242,PDTC and TAK-242+PDTC can effectively inhibit the expression of related deposition proteins on MCs to varying degrees.The results of the ROS experiment showed that compared with the normal exosomes group,the ROS levels of MCs in the HG exosomes group were significantly increased;compared with the HG exosomes group,BBR,TAK-242,PDTC and TAK-242+PDTC Both can effectively down-regulate the ROS intensity of MCs.Conclusion1.HG induces functional damage of MCs in podocyte-derived exosomes.2.BBR can improve the damage of HG-induced podocyte-derived exosomes to MCs.3.BBR may improve the functional impairment of MCs by regulating the TLR4/NF-κB/NLRP3 inflammasome signaling pathway.