| Background and objectiveThe prevalence of diabetic nephropathy(Diabetic kidney disease,DKD)is about 20%～40%,which is the main complication and death cause of diabetes mellitus.In recent years,with the rapid growth of the number of patients with diabetes,the prevalence of DKD has increased year by year,and has become the first cause of end-stage renal disease(ESRD)in some countries or regions.The pathogenesis of DKD is complex,the present study results show that a variety of factors,hemodynamic disorders and metabolic changes of the inflammatory response mechanism,cytokines,oxidative stress,genetic factors,kinin system and autophagy have played a very important role.At present,due to limited treatment,failed to effectively curb the occurrence and development of DKD,the DKD into the ESRD stage to the patient,family and government to bring heavy medical and economic burden.Therefore,it is urgent to study the pathogenesis of DKD and search for effective intervention targets.The relationship between diabetic nephropathy and micro inflammation is increasingly prominent.Innate immunity is the main driving factor of diabetic nephropathy with respect to adaptive immunity.Since 1991,Cavallo et al.Found for the first time that the serum of patients with 2TDM had inflammatory factors IL-1,IL-2,IL-4,IL-6,interferon,TNF-,and then opened the study of the inflammatory state of DKD.The main process of this immune response in DKD is:in high glucose stimulation,monocyte chemoattractant protein(monocyte chemotactic protein,MCP-1),formation of intercellular adhesion molecule(intercellular adhesion,molecular-1,ICAM-1,B),resulting in a large number of macrophages and T lymphocytes,renal cells and other inflammatory cells involved in this through the process,MAPK,I,JAK-STAT K B a variety of ways,by common NF-kappa B pathway of chemokines,inflammatory cytokines and adhesion factor activation,resulting in a pro infl ammatory effect,resulting in this process continued to go on,resulting in local kidney hyperplasia and sclerosis keeps happening.These classic inflammatory reactions promote the infiltration of mononuclear cells and lymphocytes,thereby activating and secreting harmful factors,such as inflammatory cytokines and reactive oxygen species.This leukocyte activity enhances the inflammatory response and promotes the development of cellular injury and fibrosis.Research suggests that NLRP3 inflammasome pathway,IL-1 beta,IL-18 inflammation effect factor related to inflammation factors at multiple levels affect diabetic nephropathy microinflammatory state.As one of the promoter of renal fibrosis,micro inflammation promotes the progression of diabetic nephropathy.As the NLRP3 inflammasome and micro inflammation is closely related to the multicomponent protein complex,its importance in the study of the increasingly prominent innate immune related diseases in recent years,inflammation mediated plays an important role in the pathological development of diabetic nephropathy.In vivo experiment of proximal renal tubular epithelial cells showed more proximal tubule in diabetic nephropathy development plays a crucial role in the role,which is mainly due to the proximal tubular epithelial cells in inflammatory and fibrosis.This pathological progress begins with metabolic abnormalities,such as urinary protein,high glucose,advanced glycation end products and intermediates,angiotensin II and other metabolic substrates.Hyperglycemia is the most important metabolic disorder of diabetes,and is also an important pathogenic factor of diabetic nephropathy.High glucose can promote PTECs production of proinflammatory cytokines and chemokines expression via MAPK and PKC signaling pathways,such as IL-1,CCL-2,I CAM-1,MCP-1,macrophage inflammatory protein-3a(macrophage inflammatory protein-3a,MIP-3a).The abnormal metabolism through the activation of a series of intracellular signal pathway,promote renal proximal tubular epithelial cells secrete proinflamatory and profibrotic molecules and epithelial cells to mesenchymal cells mesenchymal transdifferentiation,caused by renal tubular interstitial fibrosis and renal function in interstitial inflammation,accelerate the progress of diabetic nephropathy disease.The theory of traditional Chinese medicine for the pathogenesis of diabetic nephropathy is mainly manifested in the pathogenesis of deficiency of Qi,phlegm,blood stasis and toxin.Qi deficiency is the main factor leading to low and chronic inflammation.Astragalus is commonly used in Chinese herbal tonics,with Qi solid form,diuresis detoxication,pus and heal sore muscle function,commonly used in the treatment of qi asthenia,chronic nephritis,proteinuria and diabetes.In the research of many kinds of Chinese patent medicine and single drug,Astragalus has been proved to have good anti-inflammatory effect by more and more clinical practice and basic research.IV(astragaloside,AS-IV)is an important component of the pharmacological activity of Radix Astragali,and also an indispensable component of the quality evaluation of Radix Astragali preparation.And modern clinical research in vitro and in vivo experiments showed that AS-IV on immune function disorders,inflammation,etc.can improve the effect of regulation,and the state and DKD micro inflammation in the etiology and mechanism are closely linked,some research found that AS-IV can reduce the apoptosis of podocytes induced by high glucose against oxidative stress,thereby improving the diabetic renal injury.We conclude that AS-IV is able to slow the progression of diabetic nephropathy by reducing the inflammatory state.Therefore,in this study,we investigated the role of AS-IV in the treatment of diabetic nephropathy by NLRP3 in vitro and discussed the molecular mechanism of DKD in order to provide a scientific basis for the treatment of diabetic nephropathy.Astragal oside IV intervention on NLRP3 pathway activation induced by high glucose in NRK-52E cells(Part 1)ObjectiveAstragaloside IV intervention on NLRP3 pathway activation induced by high glucose in NRK-52E cells.MethodsNRK-52E cells were stimulated with high concentration of glucose(5.5-65mM)and different time(0-48h)to observe the effects of NRK-52E on the proliferation and proliferation of the cells.Cell viability was detected by CCK8 assay.Detection of the expression of Western core protein NLRP3 in inflammatory body of NLRP3 by Blot.Different doses of astragaloside IV(10-80 μM)were used to culture NRK-52E cells without stimulation of 24h,and cell viability was detected by CCK8 assay.The experiment was divided into 6 groups:normal control group(NG),osmotic pressure control group(30 mM L-Glucose),high glucose control group(HG),AS-IV low,medium and high dose group(10,20,40μM AS-IV).Westernblot method was used to detect the expression of apoptosis associated protein ASC,cysteine aspartate kinase precursor Pro-caspase-1 and Caspase-1 P10.Using a-Tubulin reference.ResultsCCK-8 results showed that high glucose has inhibitory effect on cell viability in a concentration dependent manner;the control group compared with normal glucose,high glucose group 25mM,45mM and 65mM concentration have significant difference between the high glucose group(P<0.05);high glucose group 45mM concentration and 65mM concentration have significant difference(P<0.05).45mM stimulated by high glucose 24h,cell viability decreased to 85.1%±0.1%g(P<0.05),65mM stimulated by high glucose 24h,cell viability decreased to 58.2±0.1%g(P<0.01).According to the results of CCK-8,the maximal stimulation concentration of 45mM was used as the experimental condition for the next experiment.The expression of NLRP3 protein in NRK-52E cells increased with the increase of high glucose concentration,and reached the highest value under 35mM(7.44±4.23 g,P<0.05).At different time points,the expression of NLRP3 was increased to the highest level(3.50±0.81g,P<0.05),and then decreased gradually,but there was significant difference compared with the control group(9h).The expression of ASC protein was also increased with the increase of high glucose concentration,and the expression level was the highest(3.80±1.09 g,P<0.05)when the high concentration of glucose was 25mM.High glucose stimulated time was the highest expression of 9h,and then decreased.Caspase-1 reached the highest level under the condition of high concentration of 25mM(3.70±1.23g P<0.05).High glucose stimulated 9h expression to the highest level(compared with Oh,an increase of 4.14±1.81g,P<0.05),and gradually decreased,but compared with the control group,there were significant differences.AS-IV can dose dependently downregulated expression of high glucose induced NLRP3 protein,including AS-IV(20mM)and high dose(40mM)and high glucose group compared significant difference(P<0.05),AS-IV low dose group(10mM)and high glucose group compared with no significant difference(P>0.05).AS-IV can dose adjustment to lower the high glucose induced expression of ASC protein,including AS-IV(20 M)and high dose(40mM)had significant differences compared with high glucose group(P<0.05),AS-IV low dose group(10mM)had no significant difference compared with the high glucose group(P>0.05).AS-IV can adjust the dose to downregulation of pro-caspase-1 induced by high glucose,caspase-1 expression level of P10 protein,including AS-IV(20mM)and high dose(40mM)compared with the high glucose group,showed that there were significant differences(P<0.05),AS-IV low dose group(10mM)and high glucose group compared with no significant difference(P>0.05).ConclusionThe micro inflammatory state in NRK-52E cells induced by high glucose in NLRP3 inflammasome activation;AS-IV can inhibit high glucose induced expression of NLRP3 inflammasome increased,the molecular mechanism is through down-regulation of NLRP3/Caspase-1 signaling pathway,inhibit the inflammatory reaction of renal tubular function.Inhibitory effect of astragal oside IV on DKD micro inflammation(Part 2)ObjectiveTo observe the inhibitory effect of AS-IV on the micro inflammatory state of NRK-52E cells induced by high glucose.MethodsTo observe the effect of concentration gradient AS-IV(10-80μM)on the viability of NRK-52E cells.Cell viability was detected by CCK8 assay.NRK-52E cells were stimulated with different concentrations of high glucose(5.5-65mM)and different time(0-48h)to detect the expression of inflammatory factor pro-IL-1β,Western,and the expression of IL-1 1β.The experiment was divided into 6 groups:normal control group(NG),osmotic pressure control group(30 mM L-Glucose),high glucose control group(HG),AS-IV low,medium and high dose group(10,20,40.M AS-IV).Western Blot method was used to examine the expression of inflammatory factor pro-IL-1β,IL-1β,and the expression of inflammatory factor IL-18 was detected by ELISA assay.Using a-Tubulin reference.ResultsWith the increase of high glucose concentration,the expression of pro-IL-1 β and IL-1 β in NRK-52E cells increased gradually,and reached the highest level under the condition of high concentration of 20mM(P<0.05).High glucose stimulation at different time points can be seen in the 9h when the expression of pro-IL-1β,IL-1β reached the highest level(P<0.05),and gradually decreased,but compared with the control group,there were significant differences.AS-IV can adjust the dose to downregulation of pro-IL-1β induced by high glucose,IL-1 0 expression level,the IL-1β,AS-IV(20μM)and high dose(40 mM)had significant differences compared with high glucose group(P<0.05),AS-IV low dose group(10 u M)with no significant difference with the high glucose group(P>0.05).The results of ELISA showed that the concentration of IL-18 in cell culture solution was significantly higher than that in normal glucose control group after high glucose stimulation,which was dose dependent and reached the highest level under 25mM(11.70±2.23g,P<0.05).High glucose stimulation at different time points can be seen in the 9h when the concentration of IL-18 reached the highest level(3.14 ± 1.81g,P<0.05),and gradually decreased,but compared with the control group,there were significant differences.To give 20 μM dose of AS-IV significantly decreased the expression of IL-18 induced by high glucose level,there was significant difference compared with the high glucose group(P>0.05);AS-IV can adjust the dose to cut high glucose induced expression of IL-18,including AS-IV(20 μM),Gao Jiliang(40 μM)was significant compared with high glucose group(P<0.05),AS-IV low dose group(10 μM)no significant difference compared with the high glucose group(P>0.05).ConclusionAS-IV can inhibit NRK-52E cells induced by high glucose and micro inflammation,its molecular mechanism is through inhibition of inflammatory activity precursor pro-IL-1β activation pathway,inhibition of inflammation factor IL-1γ and IL-18 expression,and then inhibit the DKD micro inflammatory reaction.Effects of astragaloside Ⅳ on anti fibrosis in high glucose induced NRK-52E cellsObjective:To observe the effect of AS-IV on high glucose induced NRK-52E cell fibrosis.Methods:NRK-52E cells were stimulated with high concentration of glucose(5.5-65mM)and different time(0-48h)to observe the effect of concentration gradient AS-IV(10-80 μM)on the viability of NRK-52E cells.Cell viability was detected by CCK8 assay.The experiment was divided into 5 groups:normal control group(NG),high glucose control group(HG),AS-IV low,medium and high dose group(10,20,40 μM AS-Ⅳ).The expression of Western Blot ECM Fibronectin,the main component of the detection of fibronectin type IV collagen Type IV Collagen.Using a-Tubulin reference.Results:Western blot showed that 24h NRK-52E cells cultured in high glucose environment,can induce the synthesis of cell Fibronectin increased significantly,compared with normal glucose control group,with significant difference(P<0.05);elevated levels of Fibronectin protein AS-IV inhibit glucose induced,and in a concentration dependent manner,AS-IV(20 μM),high dose(40 μM)had significant differences compared with high glucose group(P<0.05),AS-IV low dose group(10 μM)no significant difference compared with the high glucose group(P>0.05).24h NRK-52E cells cultured in high glucose environment,can induce the expression of IV Collagen cells increased significantly,and the normal glucose control group compared with significant difference(P<0.05);AS-IV(10 μM),middle(20 μM)and high dose(40 μM)had significant differences compared with high glucose group(P<0.05);low dose group(10 μ M)had significant difference compared with the normal control group(P<0.05).Conclusion:AS-IV down regulate the expression of Fibronectin and Type IV Collagen in NRK-52E cells induced by high glucose,and the mechanism related to the NLRP3 inflammatory pathway,which proves the anti fibrosis effect of AS-IV. |
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