Salvianolic Acid-B Regulates Pyroptosis By The Nrf2 In Acute Kidney Injury

Acute kidney injury(AKI)is a common kidney critical disease characterized by rapid(hours to days)decline in renal function,which can lead to multiple organ failure.Currently there is no specific treatment for AKI,so it is particularly important to explore the mechanism of AKI and to find new therapeutic targets.The main pathological feature of AKI is the death of renal tubular epithelial cells,which induces subsequent immune inflammatory responses and aggravates kidney damage.Therefore,inhibition of cell death in AKI may be a key target for reversing AKI pathological damage.Pyroptosis,a novel form of cell death dependent on Caspase-1/11,develops faster than apoptosis and is accompanied by the release of a large number of pro-inflammatory factors,which plays an important role in promoting the development of renal inflammatory diseases.Therefore,exploring the role of cell death in acute kidney injury is helpful to reveal the pathogenesis of acute kidney injury.In short,exploring the role of cell death in acute kidney injury is helpful to reveal the pathogenesis of acute kidney injury.Traditional Chinese medicine and its active ingredients have unique advantages in the prevention and treatment of acute kidney injury.Salvianolic acid B(SalB)is the main water-soluble active ingredient of Salvia miltiorrhiza Bunge.It has strong anti-oxidation effect and has acute and chronic kidney damage.Protective effects.Based on this,we intend to study the key link of cell coke in acute kidney injury,and salvianolic acid B(SalB)effectively inhibit cell coke,improve acute kidney injury,and expand the pharmacological mechanism of salvianolic acid B on acute kidney injury.Clinical applications provide new ideas.Objective1.To investigate the role of cell coronation in mice with acute kidney injury induced by ischemia-reperfusion model and the improvement of pyroptosis by salvianolic acid B,and to reveal the relationship between cell death and acute kidney injury.2.To explore the regulation of salvianolic acid B on Nrf2 and pyroptosis in acute kidney injury,and to reveal the mechanism of salvianolic acid B in improving acute kidney injury.3.At the cellular level,further clarify the molecular mechanism of salvianolic acid B to protect the renal tubular epithelial cells by regulating Nrf2 inhibition of Pyroptosis.MethodsThe high-medium-low(200,100,50 mg/kg)dose group of salvianolic acid B,and the prednisone acetate group(2 mg/kg)were pre-administered for 7 days.Seven days later,the renal ischemia-reperfusion(IRI)AKI model of the mouse was established by unilateral nephrectomy and contralateral renal artery clamping.To detect 24h serum creatinine and urea nitrogen in the mouse,and to detect the morphology of mouse kidney tissue using HE staining.To reflect oxidative stress levels in vivo through detection of SOD、MDA and GSH.To express change by detection of Pyroptosis Caspase-1 using immunofluorescence and Western Blot,and to express change by detecting pathway Protein of NLRP3-Caspase-1-GSDMD and Keapl-Nrf2/HO-1,and to investigate the intervention effect of SalB on AKI from animal level.To establish a hypoxia-reoxygenation-induced renal tubular epithelial cell(HK-2)injury model and give salvianolic acid B high,medipM and low(80,40,20 μM),VX-765(50 μM)and MCC950(50 μM),using flow cytometry to detect Annexin V/PI double positive rate;simultaneous to make the determination of lactate dehydrogenase(LDH).To detect GSDMD-N using WB and investigate the effect of cell coping on hypoxia-reoxygenation injury and the intervention of salvianolic acid B.To express the change through the detection of Pyroptosis Caspase-1 using immunofluorescence and Western Blot.The pathway protein of NLRP3-Caspase-1-GSDMD and Keapl-Nrf2/HO-1 were detected to express change,confirming the molecular mechanism of protecting renal tubular epithelial cells at the cellular level.Results1.Salvianolic acid B effectively protects kidney function,improves kidney damage,inhibits inflammatory factor secretion and oxidative stress in IRI mice.Salvianolic acid B protects IRI mice by inhibiting the nuclear expression of Nrf2 and activating the Keapl-Nrf2/HO-1 antioxidant pathway,thereby inhibiting NLRP3-Caspase-1 mediated pyroptosis.2.Salvianolic acid B effectively increased the cell viability of H/R-induced HK2 cell model,inhibited the release of LDH,and improved pyroptosis.Salvianolic acid B protects H/R from HK2 cells by promoting Nrf2 nuclear expression and activating the expression of the antioxidant pathway Keap 1-Nrf2/HO-1,thereby inhibiting NLRP3-Caspase-1-GSDMD-mediated pyroptosis.3.Compared with HK2 in H/R group,salvianolic acid B can effectively increase cell viability,decrease MDA expression,and reduce the release of LDH during the pyroptosis.Intervention with Caspase-1 specific inhibitor VX-765,NLRP3 inhibitor MCC950 and salvianolic acid B,flow results VX-765 can effectively reduce the PI double positive rate of HK2 cells,Western Blot results VX-765 and MCC950 can inhibit decreased expression of Caspase-1(Caspase-1 and GSDMD),It is suggested that H/R-induced pyroptosis is dependent on NLRP3-Caspase-1 activation.After the intervention of salvianolic acid B,the flow results showed that the PI double positive rate of HK2 cells in salvianolic acid B group was significantly decreased.The results of Western Blot showed that the expression of cox protein(Caspase-1 and GSDMD)in salvianolic acid B group decreased,suggesting that salvianolic acid B inhibition of H/R induced HK2 pyroptosis.Immunofluorescence and Western Blot results showed that salvianolic acid B inhibited the activation of NLRP3 and the expression of TXNIP protein,promoted the nuclear expression of Nrf2,decreased the expression of Keap1 protein,and promoted the expression of HO-1 protein,suggesting that salvianolic acid B may promote Keapl/Nrf2.The HO-1 pathway exerts an antioxidant action,inhibits the activation of NLRP3,and inhibits pyroptosis dependented by Caspase-1Conclusion1.Pyroptosis promotes oxidative imbalance and inflammatory response in acute kidney injury,aggravating the occurrence and development of diseases.Salvianolic acid B can effectively inhibit pyroptosis in acute kidney injury.2.Salvianolic acid B effectively protects kidney function,improves kidney damage,inhibits inflammatory factor secretion and oxidative stress in IRI mice.Salvianolic acid B protects IRI mice by inhibiting the nuclear expression of Nrf2 and activating the Keapl-Nrf2/HO-1 antioxidant pathway,thereby inhibiting Caspase-1 mediated pyroptosis.3.Salvianolic acid B effectively increased the cell viability of H/R induced HK2 cell model,inhibited the release of LDH,and improved coke.Salvianolic acid B protects HK2 cells induced by H/R,via accelerating the nuclear expression of Nrf2 and activating the expression of the anti-oxidation pathway Keapl-Nrf2/HO-1,thereby inhibiting Caspase-1-GSDMD-mediated pyroptosis.