Effect Of Salvianolic Acid A On Acute Renal Injury Induced By Lipopolysaccharide And Its Molecular Mechanism

Purpose:The purpose of this study was to investigate the effect of Salvianolic acid A?Salvianolic acid A,SA?,an active component of traditional Chinese medicine Salvia miltiorrhiza?Salvia miltiorrhiza Bunge?,on lipopolysaccharide?Lipopolysaccharides,LPS?induced acute renal injury?Acute renal injury,AKI?in mice and its mechanism.Methods:Animal experiment:32 male BALB/C mice?18±22g?were randomly divided into four groups,control group,LPS stimulation model group,SA+LPS treatment group,Dexamethasone?DEX?+LPS positive control group with eight mice in each group.Before the establishment of the model,by intragastric administration,the SA+LPS treatment group was given SA?60 mg/kg?for 1 hour,and the positive control group was given dexamethasone?5 mg/kg?by intraperitoneal injection for 1 hour.Except for the control group,the mice in the other groups were stimulated by intraperitoneal injection of LPS?10 mg/mL?for 18 hours,and the mice were anesthetized with 10%chloral hydrate18 hours later.After the kidney was embedded in paraffin and sectioned,the renal tubular injury was observed by hematoxylin-eosi?H&E?staining,the accumulation of macrophages in the injured site was observed by immunohistochemical method,and the levels of Scr and BUN were detected by serum creatinine?Scr?and blood urea nitrogen?BUN?kit.The levels of pro-inflammatory cytokines,tumor necrosis factor-??TNF-??,and mouse interleukin-6?IL-6?in serum were detected by Enzyme-linked immunosorbent assay?ELISA?.Cell experiment:the optimal concentration of SA was screened by the MTT method through the culture of macrophage line RAW264.7 in vitro.The cells were divided into the control group,LPS-stimulated model group,SA+LPS treatment group,and SA alone group.BThe SA+LPS group and SA group were pre-threaten with SA?10?M?1 hour,LPS?1?g/m L?for 18 hours,and the levels of TNF-?and IL-6 release in the cell supernatant were detected.Western blotting was used to detect inflammatory mediators cyclooxygenase-2?Cyclooxygenase-2,COX-2?and inducible nitric oxide synthase?Inducible nitric oxide synthase,iNOS?,inflammation-related signaling pathway Toll-like receptor 4?Toll-like receptor-4,TLR4?,medulla differentiation factor?Medullary differentiation factor,MyD88?,endoplasmic reticulum stress-related proteins p-PERK,CHOP,p-ElF2?,ElF2?,and mitogen-activated protein kinase?MAPK?signal pathway protein expression.Moreover,the release of nitric oxide?NO?,reactive oxygen species?ROS?,and intracellular Ca²⁺were detected by flow cytometry,and the nuclear localization of NF-?B/p65 was examined by immunofluorescence.The relationship between SA,LPS,and TLR4 binding sites was further observed by bioinformatic analysis.Results:In the in vivo experiments,the histological changes of kidney and the injury of renal tubules were observed in the LPS-induced AKI mice model.Compared with the control group,the glomeruli in the model group were atrophied and vacuolated.As expected,both SA and DEX could significantly reduce renal injury induced by LPS in model mice.The renal function was identified by serum creatinine?Scr?and blood urea nitrogen?BUN?kit,and the results were consistent with H&E staining.The levels of Scr and BUN were significantly decreased in both SA and DEX groups?p<0.01?.Besides,SA could significantly inhibit the release of inflammatory cytokines TNF-?and IL-6 in the serum of mice with renal injury?p<0.01?.The results of immunohistochemistry showed that the accumulation of F4/80 positive macrophages in the AKI group was significantly increased,while both SA and DEX reduced the infiltration of F4/80 positive macrophages.In conclusion,SA significantly ameliorates LPS induced renal injury by inhibiting glomerular atrophy and reducing the levels of Scr and BUN.Interestingly,SA could significantly reduce the release of serum inflammatory cytokines and block the inflammatory infiltration of macrophages in the injured renal tissue.In the in vitro study,the optimal concentration of SA to macrophages was detected by the MTT method.The MTT results showed there was no cytotoxicity when the concentration of SA was 10?M.In the follow-up study,the production of nitrite was detected by the Griess kit and NO flow fluorescence probe DAF-FM.These results proved that LPS stimulated the release of NO in macrophage RAW264.7,while SA could significantly decrease the release of NO induced by LPS.Western blot results revealed that SA could significantly inhibit the expression of COX-2 and iNOS in macrophages provoked by LPS?p<0.01?,and restrain the release of pro-inflammatory cytokines TNF-?and IL-6 induced by LPS in macrophages supernatant,which was consistent with the results of in vivo experiments.Western blot results showed that SA could significantly inhibit the protein expression of LPS-activated TLR4/MyD88 pathway in RAW264.7 cells?p<0.01?.Also,silencing TLR4 can significantly reduce the release of inflammatory cytokines TNF-?and IL-6,which suggests the regulatory role of TLR4 in the inflammatory process.Endoplasmic reticulum stress is associated with many acute diseases.In current studies,Ca²⁺overloading and PERK-EIF2?-CHOP pathway are associated with LPS-induced endoplasmic reticulum stress response,and SA pretreatment can reverse these phenomena.Furthermore,oxidative stress is linked with the development of acute renal injury,while SA and ROS scavenger NAC can reduce LPS-induced ROS production.Additionally,Ca²⁺chelator BAPTA-AM and reactive oxygen scavenger NAC could inhibit the release of inflammatory cytokines in macrophages induced by LPS.LPS could initiate the MAPK pathway in macrophages while SA could significantly repress the phosphorylation of JNK1/2?p<0.01?but had no significant effect on ERK1/2 and p38.Immunofluorescence results revealed that SA could significantly inhibit the translocation of NF-?B/p65 to the nucleus induced by LPS.Docking analysis showed that the binding sites of SA and TLR4competitively inhibited the stimulation with LPS,consequently inhibiting the activation of TLR4 in the LPS-induced inflammatory signaling pathway the occurrence of inflammation.Conclusion:Based on the above research background,SA may have a potential therapeutic effect modulating the inflammatory signaling pathway of AKI.In summary,SA may be used as a potential therapeutic agent for clinical prevention and treatment of AKI in the future.