The Role Of NADPH Oxidase In Acute Pancreatitis-related Kidney Injury In Hyperlipidemic Rats

Part 1 Pathological changes of pancreas and kidney and changes of NOX protein level in kidney tissue in rats with acute pancreatitis with hyperlipidemiaObjective:to explore the injury of pancreatic tissue and kidney tissue in rats with hyperlipidemic acute pancreatitis,and to explore whether NOX protein in kidney tissue plays a role in aggravating renal tissue injury.Methods: 8week-old SPF SD rats were randomly divided into four groups: normal control group(N-CON group),non-hyperlipidemic pancreatitis group(NLAP group),hyperlipidemic control group(H-CON group)and hyperlipidemic pancreatitis group(HLAP group).According to the sampling time,the hyperlipidemic pancreatitis group was subdivided into three subgroups: HLAP 12 h,HLAP 24 h and HLAP 48 h,with 8rats in each group.The normal group was fed for 14 days,and the hyperlipidemia model was induced by continuous intraperitoneal injection of 0.25g/kg for 14 days.After 14 days,the rats in the two groups were induced with acute pancreatitis by intraperitoneal injection of L-Arg twice(2.5mg/kg/,with an interval of 1 hour).After the completion of the model,the rats were fasted and watered,and the samples were killed under anesthesia at the corresponding time point.Venous blood was collected from the inferior vena cava,and appropriate amount of pancreatic tissue and kidney tissue were fixed and cryopreserved.Serum amylase level(AMY),liver function related serum index level(ALT,AST),renal function related serum index level(Urea,Cr)and blood lipid related index level(TG,TC,FFA)were detected by automatic biochemical instrument.The fixed pancreatic tissue and kidney tissue were embedded and sectioned and stained with HE.The histopathological changes were observed and scored under light microscope.The levels of malondialdehyde((MDA))and superoxide dismutase(SOD)(SOD)in serum,pancreas and kidney were detected by kit.The protein expression levels of NOX4 in kidney tissue of rats in HLAP group were detected by Western blot method after 12 h,24h and 48 h.Results: after continuous intraperitoneal injection of Pmur407 for 14 days,the levels of serum TG,TC and FFA increased significantly.In addition,the results of liver and kidney function showed that Pmur407 alone had no significant effect on serum ALT,AST,Urea and Cr.After the acute pancreatitis model was induced by L-Arg,the increase of serum amylase,obvious damage of liver and kidney function,obvious hemorrhage and necrosis of pancreatic tissue and renal tubular epithelial cell swelling,brush margin shedding and tube formation were observed in both high fat group and non-high fat group.Compared with the hyperlipidemic acute pancreatitis group and the non-hyperlipidemic acute pancreatitis group,the serum hepatic and renal function indexes of rats in the HLAP group increased more significantly,and the pathological damage of pancreatic tissue and kidney tissue was more serious than that in the nonhyperlipidemic group.The results of MDA and SOD in serum,pancreatic tissue and kidney tissue were similar.After the acute pancreatitis model was induced,the levels of MDA and SOD in serum,pancreatic tissue and kidney tissue increased significantly in hyperlipidemic acute pancreatitis group and non-hyperlipidemic acute pancreatitis group,and the levels of MDA and SOD in serum,pancreatic tissue and kidney tissue increased more significantly in hyperlipidemic acute pancreatitis group.The results of Western blot showed that the expression levels of NOX4 in kidney tissue of HLAP group were higher than those of hyperlipidemia control group at each time point,and reached the peak after 24 hours of modeling.Conclusion: 1.The function and structure of kidney in rats with hyperlipidemic acute pancreatitis can be significantly damaged,and the injury is more severe than that in rats with non-hyperlipidemic acute pancreatitis.high concentration of FFA in hyperlipidemia may aggravate the damage of renal structure and function in hyperlipidemic acute pancreatitis by activating the expression of NOX protein in renal tissue.Part 2 The role and mechanism of NOX in renal injury induced by acute pancreatitis in hyperlipidemic ratsObjective: to determine the role and mechanism of NOX protein in renal injury in hyperlipidemic rats with acute pancreatitis.Methods: SPF male SD rats were randomly divided into six groups: normal control group(N-CON group),non-hyperlipidemic pancreatitis group(NLAP group),hyperlipidemic control group(H-CON group),hyperlipidemic pancreatitis group(HLAP group)and hyperlipidemic Apocynin intervention group(HAPO group).The appropriate intervention dose was evaluated and screened out after the intervention of Apocynin with three doses of 25,50 and 100mg/kg.The specific modeling methods are as follows: N-CON group,NLAP group,H-CON group and HLAP group were the same as the first part.The rats in HAPO group were subcutaneously injected with Apocynin one hour before intraperitoneal injection of Apocynin(the dose is subject to prescreening),and the other operations were the same as those in NLAP and HLAP groups.After successful modeling,the rats were fasted and watered.24 hours later,the rats were killed under isoflurane anesthesia.Venous blood was collected from the inferior vena cava,and appropriate amount of pancreatic tissue and kidney tissue were fixed and frozen respectively.Serum amylase level(AMY),liver function related serum index level(ALT,AST),renal function related serum index level(Urea,Cr)and blood lipid related index level(TG,TC,FFA)were detected by automatic biochemical instrument.The fixed pancreas and kidney tissues were embedded and sectioned and stained with HE.The histopathological changes were observed under light microscope and the pathological scores were evaluated.In addition,a small amount of renal tissue was fixed and sliced in the fixed solution of electron microscope,and the ultrastructural changes were observed under transmission electron microscope.The levels of MDA and SOD in renal tissue were detected by kit,and the level of ROS in renal cells was detected by ROS fluorescent probe-DHE.The protein expressions of NOX4,p-Akt,GSK-3 β,NF-κ B,TNF-α,MPO and CD68 in kidney tissue were detected by Western blot,immunohistochemical staining and immunofluorescence staining.TUNEL staining was used to detect the level of apoptosis in kidney.Results: preconditioning with Apocynin at the dose of 50mg/kg could effectively improve the level of serum AMY and improve the indexes of serum liver and kidney function in rats with hyperlipidemic acute pancreatitis,but had no significant effect on the levels of serum TG,TC and FFA.Pancreatic tissue and kidney tissue showed obvious pathological damage in hyperlipidemic acute pancreatitis,and the injury in hyperlipidemic group was more serious.Apocynin pretreatment can effectively improve the renal injury in hyperlipidemic acute pancreatitis rats.In addition,in the hyperlipidemic acute pancreatitis group,the mitochondria and endoplasmic reticulum of renal tubular epithelial cells were obviously swollen,the brush margin was damaged or even part of the brush edge was broken and disappeared,and some of the severe cells were on the verge of death.After Apocynin pretreatment,the expansion of endoplasmic reticulum and mitochondria and the injury of brush margin were alleviated in HAPO group.The renal tissue of rats with hyperlipidemic acute pancreatitis had a higher level of ROS,and Apocynin could reduce the level of ROS in the kidney.After administration of NOX inhibitor Apocynin,the levels of NOX4 in kidney tissue were significantly decreased,the activation of proteins related to downstream Akt/GSK-3β pathway was also significantly inhibited,and renal cell apoptosis was significantly reduced.Conclusion: 1.NOX can aggravate renal injury in rats with hyperlipidemic acute pancreatitis by regulating the release of ROS and activating Akt/GSK-3β pathway.2.Apocynin can improve renal injury in rats with hyperlipidemic acute pancreatitis by inhibiting the expression of NOX protein,reducing the release of ROS and inhibiting the activation of Akt/GSK-3β.Part 3 Study on the mechanism of NOX in HK-2 cell injury induced by FFAObjective: to investigate whether high concentration of FFA can damage HK-2 cells and its mechanism.Methods: HK-2 cells were treated with different concentrations of palmitic acid to screen the appropriate concentration.After the concentration of PA was determined,no dose of Apocynin was designed to intervene,and the appropriate dose of Apocynin was selected.The cells were then divided into four groups: control group,PA intervention group,PA+Apocynin intervention group and Apocynin control group,in which the control group was not treated,while the PA+Apocynin intervention group was treated with selected dose of Apocynin for 1 hour and then treated with corresponding dose of PA for 24 hours,and the Apocynin control group was treated with selected PA and Apocynin for 24 hours,respectively.The level of apoptosis was detected by flow cytometry,the level of IL-1 β and TNF-α in the supernatant of each group was detected by ELISA kit,the level of ROS was detected by fluorescent probe,and the level of NOX4 and the expression of p-Akt and GSK-3 β were detected by immunofluorescence staining.Results: 0.5m M treated with PA for 24 hours could cause a large number of HK-2 cell death,and intervention with 0.4m M ’s Apocynin 1 hour before PA treatment could effectively alleviate HK-2 cell death induced by PA.The results of flow cytometry showed that there was obvious apoptosis in HK-2 cells treated with 0.5 m M PA alone,and the apoptosis was significantly improved in combination with PA+Apocynin group.The levels of IL-1 β and TNF-α in the supernatant of HK-2 cells treated with 0.5 m M PA were significantly higher than those in the control group.After the administration of Apocynin,the levels of IL-1 β and TNF-α in the supernatant decreased significantly.The ROS level of HK-2 cells in PA group was significantly higher than that in the control group,and Apocynin pretreatment could significantly decrease the ROS level.Immunofluorescence staining showed that the positive levels of NOX4 in PA group were significantly higher than those in control group,and the expression levels of pAkt and GSK-3β in HK-2 cells were also significantly increased.Apocynin pretreatment with 0.4m M could effectively inhibit the expression of NOX4 proteins,and inhibit the expression levels of downstream p-Akt and GSK-3 β proteins.Conclusion: 1.High concentration of free fatty acids can cause damage to human renal tubular epithelial cells,affect their cell viability and promote cell apoptosis.two。.Free fatty acids can activate the NOX,of HK-2 cells to produce a large number of ROS,to activate the downstream Akt/GSK-3 β pathway,promote the release of inflammatory mediators and apoptosis,thus damaging HK-2 cells.3.Apocynin treatment can inhibit NOX protein in HK-2 cells,reduce the release of ROS,block the activation of downstream pathways,and thus protect cells from FFA-induced injury.