The Mechanism Of TLR4/NF-κB Signaling Pathway Mediated Crush Syndrome-related Acute Kidney Injury In Rats

Objective:Crush syndrome refers to muscle or ischemic damage caused by heavy weights of muscle-rich parts after being compressed for a long time.A series of systemic reactions characterized by acidosis.Crush syndrome not only aggravates renal ischemic damage under post-traumatic systemic stress,but also forms an acidic methaemoglobin cast that blocks the renal tubules.The myoglobin is quickly released into the blood and penetrates into the kidney with blood circulation and that eventually lead to acute kidney injury.Early treatment of crush syndrome is the key to reducing mortality and disability.At present,and there is no recognized and reasonable scheme.This topic aims to provide experimental and theoretical basis for clinical prevention and treatment by studying and exploring the mechanism of TLR4/NF-κB signaling pathway in crush syndrome-related acute kidney injury.Methods: This study is divided into four parts: Part 1: Establish rats crush injury model,which compressed for 16 h by crush strength at 3kg,and divide into 4 groups(0h,6h,12 h,24h)according to the different decompression treatment time.Observe the kidney tissue pathological HE staining,and TLR4,TLR4 m RNA,CK18,and p65 expressions were measured.Serum IL-6,TNF-ɑ,and kidney injury-related indicators(urea nitrogen,creatinine,and blood potassium)were detected.Part 2: Normal rat renal tubular epithelial cells NRK-52 E cells were added to normal rat serum(1,2 ml),squeezed rat serum(1,2 ml),the optimal serum concentration was selected based on TLR4 expression and then grouped: blank control group,control serum group and crush serum group.Observe renal cell morphology changes,detect cell proliferation / toxicity,cell cycle and apoptosis,detect TLR4,p65 localized expression and nuclear / plasma phosphorylation and total p65 expression,and detect serum IL-6 and TNF-ɑ expression levels.Part3:Design and synthesize and package TLR4 si RNA lentivirus,select appropriate MOI to divide NRK-52 E cells into 6 groups: control group,control serum group,crush serum group,crush serum + si RNA NC group,crush serum + TLR4 si RNA group,crush serum + TAK-242 group.Observe renal cell morphology changes,detect cell proliferation / toxicity,cell cycle and apoptosis,detect TLR4,P65 localized expression,nuclear / plasma phosphorylation and total p65 expression,and detect serum IL-6,TNF-ɑ expression levels.Part IV: Establish rats crush injury model and divide into 3 groups: control group,crush group,crush + TAK-242 group.Observe rat kidney tissue and muscle tissue pathological HE staining,detect TLR4 protein,TLR4 m RNA,CK18 and p65 expression,respectively,and detect serum IL-6,TNF-ɑ and kidney injury related indicators(urea nitrogen,creatinine,blood potassium).Result: 1.Compared with the control group,the renal tubular epithelial cells in the crush group were disorderly arranged and appeared vacuole,and the cell destruction was most significant in the 12 h decompression group;urea nitrogen,creatinine,blood potassium,TLR4 protein,TLR4 m RNA,p65,The expressions of IL-6 and TNF-ɑ increased significantly,and the expression levels in the 12 h decompression group were the most significant.There was no significant difference in p65 expression between the crush group and the control group in immunohistochemistry.The expressions of TLR4 and P65 in immunofluorescence staining were significantly higher than those in the control group,and there was no significant difference between the different crush groups.2.Select 5% final concentration for subsequent experiments.There was no significant difference in cell morphology of each group under light microscope.The proportion of G1 phase in the squeezed serum group increased,and G1 phase block may occur.Compared with the blank control group and the control serum group,the expression of TLR4 protein,TLR4 m RNA,active oxygen content of cells,and the expression of phosphorylated p65,IL-6 and TNF-α in the nucleus of the squeezed serum group were significantly increased,and the cell proliferation rate was significantly reduced;There was no significant difference between the control group and the control serum group.There was no significant difference in the expression of total NF-kappa B p65 in the three groups of cytoplasm.3.Select MOI = 30 for subsequent experiments.There was no significant difference in cell morphology of each group under light microscope.Compared with the blank control group,control serum group,crush serum + TLR4 si RNA group and crush serum + TAK-242 group,the proliferation rate of crush serum group and crush serum + si RNA NC group was significantly lower,and the proportion of G1 phase was significantly lower.Increased intracellular reactive oxygen content increased,and the expression of TLR4 protein,TLR4 m RNA,and phosphorylated p65,IL-6,and TNF-α in the nucleus increased significantly.Compared with the blank control group and control serum group,crush serum + TLR4 The proliferation rate of the si RNA group and the crush serum + TAK-242 group was slightly reduced.There was no significant difference in the proportion of G1 phase and intracellular reactive oxygen species.The expression of TLR4 protein,TLR4 m RNA,and phosphorylated p65,IL-6,and TNF-α in the nucleus were not significant difference.There was no significant difference in the expression of total NF-kappa B p65 in each group in the cytoplasm.4.Compared with the control group,the kidney and muscle tissue of the crush group were loose,irregular,and obvious vacuoles were produced.The crush + TAK-242 group had less damage than the crush group.The levels of TLR4 m RNA,TLR4 protein,p65,IL-6,and TNF-α were significantly increased in the crush group,and TLR4 m RNA,TLR4 protein,p65,IL-6,and TNF-α in the crush + TAK-242 group were significantly increased.The expression level was significantly lower than that in the crush group and slightly higher than the control group.Conclusion: 1.Crush injury in rats induces acute kidney injury,muscle tissue destruction,and eventually leads to crush syndrome,which causes the body tissue to release a large number of inflammatory factors.2.The TLR4/NF-κB signaling pathway mediates the occurrence of crush syndrome-related acute kidney injury in rats.3.By inhibiting the expression of TLR4,it can effectively reduce the severity of crush syndrome-related acute kidney injury in rats.