Abelmoschus Manihot Protects Renal Tubular Epithelial Cell Via Regulating ERK1/2-NLRP3 Inflammasome

Background/AimRenal tubular cells maintain normal renal function and are the most important part of kidney.When exposed to stimulates,Renal tubular cells show various degenerative changes and acute reversible or irreversible damage.Renal tubular repairing process,epithelial-mesenchymal trans differentiation(EMT),renal fibrosis,acute kidney injury(AKI)-chronic kidney disease(CKD)are all important to Chronic renal disease.The extent of the decline in renal function and renal tubular interstitial damage levels are closely related to each other.As the final common pathway,renal tubular injury is a major determinant of kidney disease progress.Therefore,protection of the renal tubular epithelial cells,is important for the integrity of the structure and function of kidney.However,the intervention and protection means of renal tubule cells is limited.In protecting renal tubules,Chinese herbal medicine has long been regarded as cheap and effective.Abelmoschus Manihot has long been used to treat nephritic proteinuria,and Abelmoschus Manihot capsules have been approved for class ? drugs for the treatment of chronic glomerulonephritis in China.We also found in rats,Abelmoschus Manihot can inhibits P38 signaling pathways,relieve glomerular pathological damage.Earlier studies mainly focused on the protection of glomerulus,but whether AM can protect the renal tubular epithelial cells is unclear.This study intends to establish Adriamycin induced NRK-52E cell injury model,and Adriamycin nephropathy rats model,to explore the mechanism of Adriamycin induced epithelial cell damage and the protective effect of AM.MethodsIn vitro:(1)Different concentration of Adriamycin(0,1,0.5,1.5ug/mL)were used to stimulus cells for 24h,and we use CCK8 to detect cell vitality,flow cytometry to detect apoptosis ratio;(2)Adriamycin(1?g/mL)was used to establish model of renal tubular epithelial cells apoptosis.With or without AM extract(TEA)intervention,we examined cell morphological changes,cell activity,and evaluated cell apoptosis by flow cytometry FITC/PI staining and TUNEL staining.(3).We detected reactive oxygen species(ROS)under different time.With or without TEA,with or without NAC,we evaluated the level of reactive oxygen species,Western blot shows phosphorylation levels of MAPK signaling pathways(P38 ERK and JNK),Cell activity were also observed.(4)Western blot were used to detect phosphorylation level of ERK,P38 after 0,1,3,6,9 hours of ADR incubation.(5).ERK phosphorylation and NLRP3 expression were also detected,With or without TEA,ERK inhibitor U0126,cells viability were detected after Adriamycin incubation.NLRP3 knockdown NRK-52 E cell lines,were employed to evaluated the role of NLRP3 in Adriamycin cell injury.The downstream proteins,IL-1 beta,Caspase-lwere also detected;In vivo:(1)The model were established by twice injection of Adriamycin(4 mg/kg,2 mg/kg)and left nephrectomy.With AM(1.5 g/kg/d)intervention,we also set up the negative control group(normal saline 2 ml),28 days later we executing animals and collected blood sample,urine,kidney.We use HE staining to observe rat kidney and tubular structure,Masson staining to observe collagen fibers deposition.Immunohistochemical were used to evaluate rat renal tissue NLRP3 expression and downstream effector molecule caspase-1 and IL-1 beta expression.(2)Rat serum creatinine and 24 hours urinary protein,blood albumin were also detected.ResultsIn vitro:(1)We use different concentration of Adriamycin(0,1,0.5,1.5 ?g/mL)to stimulate NRK-52E cells,CCK8 shows cell vitality gradually declined as long as the decrease in the number of cells.Flow cytometry detection shows apoptosis rate rose gradually with the increased of Adriamycin dosage;(2)AM extract(TEA)(100?g/mL)alleviate the decrease in cell number induced by Adriamycin,TEA also up regulated cell activity.Flow cytometry FITC/PI staining and TUNEL staining shows TEA protected renal tubular cells.(3)Adriamycin induced ROS production in NRK-52E.TEA can reduce the level of reactive oxygen species.NAC can protect Adriamycin induced cell injury.Western blot shows Adriamycin up regulated MAPK signaling pathways P38,ERK and JNK protein phosphorylation levels,but similar to NAC,TEA only acts on the inhibition of ERK phosphorylation.(4)Western blot detection shows Adriamycin induced ERK phosphorylation and NLRP3 expression was in a time-dependent manner,and ERK phosphorylation(1 hour)is earlier than the NLRP3 activation(6 hours).Cell activity detection showed TEA,ERK inhibitor U0126 can both reduce the apoptosis induced by Adriamycin,which means AM may inhibit ERK phosphorylation to protect cells.We use siRNA to build NLRP3 knock down NRK-52E cell lines.As shown in cell activity compared with the model group,Adriamycin intervention do less injury in the NLRP3 knock down cells.Western blot indicated that NLRP3 and its downstream protein Caspase-1,IL-1 beta activation can be inhibited by TEA and ERK inhibitor U0126,NLRP3 inhibitors INF 39.It means by inhibiting NLRP3 and upstream ERK phosphorylation,AM protected renal tubular epithelial cells.In vivo:HE staining shows renal tubular epithelial cell falls off,naked membrane and regeneration were detected in Adriamycin group(ADR).Meanwhile,ADR+TEA group has less injury than ADR group.Masson staining shows in Adriamycin group(ADR),capillary loops and Baumann's capsule adhesion.Renal tubular epithelial cell falls off,naked membrane can be found.But there are no sign of interstitial fibrosis.Renal tubular injury were significantly reduce in ADR+TEA group.Immunohistochemical shows activation of NLRP3 signal pathways,and AM reduced the NLRP3,caspase-1 protein and IL-1 beta protein expression.(2)After injection of Adriamycin+left nephrectomy,proteinuria increased,and TEA can significantly reduce the proteinuria.Serum albumin were significantly reduced in ADR+TEA group.But the serum creatinine level changes has no statistical significance.Conclusion(1)Adriamycin can induce the apoptosis of renal tubular epithelial cells.(2)Adriamycin can induce renal tubular epithelial cells via ERK1/2-NLRP3 inflammasome.(3)AM can protect renal tubular epithelial cells via inhibiting ERK mediated NLRP3 inflammasome activation.(4)In Adriamycin rat model,AM protected renal tubular via inhibiting NLRP3 inflammasome.