Exploring The Mechanism Of Baicalin On Improving Oxidative Stress And Inflammatory Response In Diabetic Kidney Disease

Section 1The mechanism of baicalin on improving oxidative stress and inflammation in diabetic kidney disease via modulating Nrf2 and MAPK signaling pathwaysObjective: To investigate the mechanism of baicalin(BAI)on oxidative stress and inflammation of diabetic kidney disease(DKD)by modulating erythroid-derived nuclear factor 2-related factor 2(Nrf2)and mitogen-activated protein kinase(MAPK)signaling pathways.Methods: The db/db mice were randomly divided into model group and BAI group,while db/m mice were used as control group.After BAI intervention for 8 weeks,the body weight,fasting blood glucose(FBG),insulin,glucose tolerance test(GTT)and insulin tolerance test(ITT)were measured.The 24-hour urine volume of the mice was recorded,and the urine microalbumin,urine and serum creatinine and blood urea nitrogen(BUN)were detected.Periodic acid–schiff(PAS)and Masson stainings were applied to estimate the histopathological changes,and the ultrastructure of glomeruli was observed by transmission electron microscope(TEM)and scanning electron microscope(SEM).Terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)staining and the detection of apoptosis-related proteins were used to assess the level of apoptosis in kidneys.Also,superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),catalase(CAT)and malondialdehyde(MDA)were examined to detect oxidative stress.The inflammatory cell infiltration was detected by immunohistochemistry,and the m RNA levels of proinflammatory cytokines involving IL-1β,IL-6,MCP-1 and TNFα were detected by Real-time quantitative polymerase chain reaction(RT-q PCR).The protein expression levels of Nrf2,HO-1 and NQO-1 were measured by Western Blot.The anti-inflammatory mechanism of BAI was clarified by renal transcriptomics approach,and the protein expression levels of p-JNK,JNK,p-P38,P38,p-Erk1/2 and Erk1/2 were detected by Western Blot for final verification.Results: Our results found that BAI effectively ameliorated diabetic conditions,proteinuria,renal histopathological changes and cell apoptosis in DKD.BAI significantly improved the kidney levels of SOD,GSH-PX and CAT,and reduced MDA level.Meanwhile,the infiltration of inflammatory cells including T-lymphocytes,T-helper cells,neutrophils and macrophages,and the m RNA levels of IL-1β,IL-6,MCP-1 and TNFαwere also obviously inhibited by BAI.Afterward,Western Blot found that BAI significantly activated Nrf2 signaling and increased the expression of downstream antioxidant enzymes HO-1 and NQO-1.Kidney transcriptomics revealed that the inhibition of MAPK signaling pathway may contribute to BAI’s anti-inflammatory activity,which has also been verified in later experiment.BAI treatment did obviously inhibit the activation of canonical proinflammatory signaling pathway MAPK family,such as Erk1/2,JNK and P38.Conclusions: In summary,our data provided evidence that BAI can treat DKD by alleviating oxidative stress and inflammation,and its underlying mechanisms were associated with the activation of Nrf2-mediated antioxidant signaling pathway and the inhibition of MAPK-mediated inflammatory signaling pathway.Section 2The mechanism of baicalin on improving oxidative stress and inflammation in podocytes via modulating PI3K/Akt signaling pathwayObjective: To explore the potential effect and molecular mechanism of BAI on improving podocyte injury model induced by palmitic acid(PA)in vitro.Methods: First,cytotoxicity experiments were performed to screen the BAI concentration used in the experiment.The podocyte marker proteins Podocin and Desmin were detected to evaluate the effect of BAI on podocyte injury,and TEM was used to evaluate apoptosis level.H2 DCFDA probe staining was used to determine the corrective effect of BAI on podocyte oxidative stress injury,and Western Blot was applied to examine the protein expression levels of Nrf2,HO-1 and NQO-1.The expression levels of proinflammatory cytokines IL-1β,IL-6,MCP-1 and TNFα in podocytes were detected by RT-q PCR.The anti-inflammatory and anti-oxidative stress effects of BAI may be associated with the regulation of PI3K/Akt signaling pathway through transcriptomics.Finally,the protein expression levels of p-Akt,Akt,p-GSK3β,GSK3β and inflammatory response-related p-JNK,JNK,p-ASK1 and ASK1 were detected by Western Blot for mechanism verification.Results: BAI intervention notably improved PA-induced podocyte injury and apoptosis,with increased Podocin expression,and decreased Desmin expression.BAI significantly inhibited the accumulation of reactive oxygen species(ROS)in podocytes,increased the protein expression levels of Nrf2,HO-1,and NQO-1,and ameliorated oxidative stress.Meanwhile,BAI intervention suppressed the expression of proinflammatory cytokines IL-1β,IL-6,MCP-1 and TNFα.Transcriptomic results showed that PI3K/Akt signaling may act as a common upstream signal for oxidative stress and inflammatory response.Western Blot showed that BAI significantly up-regulated the protein expressions of p-Akt and p-GSK3β,and down-regulated the protein expressions of inflammation-related signals p-JNK and p-ASK1.Conclusions: BAI can improve oxidative stress and inflammatory response in PA-induced podocyte injury model,thereby exerting a protective effect on podocytes.The molecular mechanism may be correlated with the activation of PI3K/Akt signaling pathway,which in turn enhances Nrf2-mediated antioxidant signaling and inhibits MAPK-mediated inflammatory signaling.