Protective Effect Of HBSP On Kidney Injury Induced By Lipopolysaccharide Through PI3K/Nrf2 Pathway

BackgroundSepsis usually refers to infection complicated with systemic inflammatory response syndrome,caused a series of pathological changes of the body,is a kind of out of control,serious and persistent,systemic inflammation,severe cases can cause multiple organ failure(Multiple Organ Failure,MOF)[1].Sepsis is a leading predisposing factor of acute kidney injury(AKI),contributing to approximately 50%of AKI cases in critically ill patients.Kidney as one of the most vulnerable organs,Once involved,it often indicates a poor prognosis.Despite the anti infection treatment and organ function support technology has made great progress,the mortality of sepsis induced acute kidney injury rate remains high,partly due to the lack of understanding of the pathogenesis of AKI.Ample evidence has put forward apoptosis and inflammation as more critical players in the pathophysiology of sepsis-induced AKI,rather than pure necrosis[2].During sepsis,inflammatory mediators derived from bacterial products as well as released by immune cells,such as lipopolysaccharide(LPS)and cytokines,could guide the immune system to fght infection and also canpotentially injure the renal function[3].LPS is the dominant cell wall molecule of Gram-negative bacteria,contributing to inflammatory responses,and could be constantly released by leukocytes at a low level throughout infection[4].Increasing proof demonstrates that LPS is related to the pathogenesis of septic AKI[5.6].Recently,LPS has been widely used as a model of experimental sepsisinduced AKI.In addition,it has been found that LPS can cause oxidative stress damage[7].Renal tubular epithelial cells are very sensitive to ischemia,hypoxia,poisoning,sepsis and other damage factors,and they are the most vulnerable targets of acute renal injury.Oxidative stress may play a key role in the injury of sepsis in renal tubular epithelial cells,excessive oxygen free radicals produced during sepsis can directly induce apoptosis of renal tubular epithelial cells by peroxidation of lipids,nucleic acids,proteins and other macromolecules,eventually lead to renal injury.Exogenous antioxidants may not reduce oxidative stress,but sometimes increase oxidative stress and cause further damage to the organism.The protective mechanism of the Nrf2(NF-E2-related factor 2)/ARE(antioxidant response element)pathway,which has an endogenous antioxidant effect,has aroused people’s attention.Physiological conditions,Nrf2 in a state of low concentration and inactive,and vulnerable to ubiquitin degradation,cell sub protein sample Kelch ECH joint protein 1(Kelch-like ECH-associated protein 1,Keap1)bind to Nrf2 and enable the Nrf2 limit in the cytoplasm,unable to enter the nucleus,its transcriptional activity is inhibited.Only when free radicals and chemical stimuli are stimulated,the oxidant or pro-electron compounds make the Keapl conformation change,or Nrf2 is directly phosphorylated.Nrf2 and Keapl uncoupling,activation of Nrf2 into the nucleus,combined with ARE,Start the expression of a series of protective gene promoter such as phase Ⅱ detoxification enzyme gene,antioxidant genes or antioxidant proteins,so as to to resist the external factors such as oxidation and chemical stimulus,restore and maintain the stability of internal environment[8].Nrf2,as a key regulator of the effective and broad-spectrum antioxidant system in cells,can greatly enhance the anti-inflammatory,antioxidant,anti chemical,and anticancer properties of the cells.When Nrf2 is absent or activity is inhibited and can not be expressed,its body protective effect is obviously weakened,and cells are vulnerable to inflammatory factors,oxidants,physical and chemical factors,carcinogens and other damage[9].Research on the protection of Nrf2 pathway on endotoxin induced AKI kidney is relatively less,research shows that in Nrf2 knockout mice,normal and induced under phase Ⅱenzymes such as glutathione S transferase(GST),NADH quinone oxidoreductase(NQOl),gamma glutamyl cysteine synthetase(gamma-GCS)significantly reduced[10].The rats with Nrf2 knockout were more likely to have inflammatory reaction,sepsis peritonitis and endotoxic shock,and the mortality was higher[11].At present,it is found that activation of the Nrf2 signaling pathway can alleviate renal damage and protect renal function in diabetic nephropathy,renal ischemia reperfusion,contrast nephropathy,and drug-induced kidney injury[12.13].Among them,PI3K/AKT pathway is more studied.PI3K is a lipoprotein kinase,which distributes in many cells in vivo.After activation of PI3K,phosphorylated 4.5-two inositol phosphate(PIP2)on the plasma membrane generates 3,4,and 5-three phosphatidylinositol phosphatidylinositol(PIP3)and activates its downstream target kinase Akt[14].Akt participates in a variety of important physiological processes,such as regulating cell proliferation and apoptosis,growth and survival.It is involved in the phosphorylation of Nrf2,only phosphorylated Nrf2 can be separated from the Keapl protein,and then from the cytoplasm into the nucleus,which is an important process of Nrf2 activation,Nrf2 activation to start downstream antioxidant-related gene transcription,play the role of body antioxidant.The erythropoietin-derived peptide is the latest red erythropoietin analog peptide,independent of the red-acting site,retains EPO activity and no EPO adverse reactions.In recent years,EPO,which is primarily used for renal anemia,has been shown to have protective effects on ischemia-reperfusion injury in the brain,heart and kidneys.A new study published in 2009 by Aoshiba[15]showed that EPO used in lipopolysaccharide(LPS)-induced endotoxin mice reduced mortality and had protective effects on liver,spleen,lymph,and lung.The multiple organ protections of EPO are related to their antioxidant effects.EPO can inhibit lipid peroxidation and enhance the activity of antioxidants such as catalase and glutathione peroxidase(GSH-PX)in vivo.PI3K/AKT pathway has been widely concerned.In 2011,Talan[16]et al compared the protective effects of EPO and HBSP like peptides on cardiac function and their effects on oxidative stress through in vitro experiments and myocardial ischemia models in rats,it was found that HBSP could reduce the infarct size,maintain the ventricular structure and Significantly increases the release threshold of oxygen free radical ROS,and has the same protective effect as EPO.However,the role of EPO and its derivatives in renal injury induced by LPS remains unclear.Considering that the pathogenesis of sepsis is due to various factors such as inflammatory mediators,apoptosis and oxidative stress,it is possible that EPO derived peptide-HBSP may have protective effects on sepsis.We applied LPS to HK-2 cells and rats to establish septic AKI cell models and animal models respectively.We tested the antioxidant activity of HBSP from in vitro and in vivo experiments,and explored the potential intracellular signal transduction mechanism.The subject is divided into three parts,in the first part we cultured human renal tubular epithelial cells(HK-2 cells)in vitro,given different intervention,to investigate the effect of erythropoietin derived peptide(HBSP)on oxidative stress and apoptosis in human renal tubular epithelial cells induced by lipopolysaccharide(LPS)and provide the preliminary basis for the prevention and treatment of endotoxin-induced renal injury by erythropoietin-derived peptide(HBSP).Part II:To explore the mechanism of HBSP fromthe inherentantioxidant pathway PI3K-Akt and Nrf2 pathway,and to provide a sufficient basis for the prevention and treatment of endotoxin-induced renal injury by HBSP.Part III:To establish a model of renal injury induced by endotoxemia in rats and further confirm the protective effect and possible mechanism of erythropoietin derived peptide(HBSP)on renal injury.This study will help us to understand the pathogenesis of acute renal injury in sepsis and may help us to find more effective prevention and treatment strategies for acute renal injury in sepsis.Part I:The effects of HBSP on oxidative stress injury of HK-2 induced by lipopolysaccharideResearch purposes1、To observe the effect of different concentrations of lipid polysaccharides on the vitality and apoptosis of human renal tubular epithelial cells.2、To observe the effects of different concentrations of erythropoietin derived peptide(HBSP)on the vitality and apoptosis of the renal tubular epithelial cells induced by lipid polysaccharide.Research method1、Human renal tubular epithelial cells(HK-2)were cultured in vitro and cultured with different concentrations of LPS(0,10,100,500,1000,10000ng/ml)for 24 hours.Cell viability was determined by MTT assay.Apoptosis rate of renal tubular epithelial cells was detected by Annexin V-FITC/PI staining and flow cytometry.Based on the above results,we select the appropriate LPS concentration for the next step.2、HK-2 cells were pretreated with different concentrations of HBSP(0,10,25,50,125ng/ml)for 2 hours,Then given 500 ng/ml LPS,MTT assay was used to measure cell viability,Annexin V-FITC/PI staining combined with flow cytometry to detect the apoptosis of renal tubular epithelial cells,to evaluate the protective effect of HBSP on cell injury induced by LPS.Based on the above results,we select the appropriate HBSP concentration for the next step.3、The cells were divided into four groups:negative control group,HBSP group,LPS group,HBSP + LPS group.HBSP + LPS group was preincubated with 50 ng/ml HBSP for 2 hours,followed by preincubation with 500 ng/ml LPS for 24 hours,and the cells were incubated in fresh medium for 24 hours.The content of ROS was measured by diacetyl dichlorofluorescein(DCFH-DA)fluorescent probe and the content of malondialdehyde(MDA)was determined by thiobarbituric acid method.Research results1、Different concentrations of LPS acted on HK-2 cells,and the cell activity(chart 1A)of each group was(84.1 ±0.7)%,(82.6± 1.5)%,(61.9± 0.6)%,(53.7± 0.8)%,(40.7± 0.9)%,(37.3± 1.9)%.Compared with the control group,the cell viability of the 100,500,1000,10000ng/ml LPS group decreased significantly,There were significant differences,with statistical significance(P<0.05).The rate of apoptosis in each group(chart 1B and 1C)was(4.87± 1.29)%,(14.01 ± 2.27)%,(39.58± 3.12)%,(51.03 ± 4.32)%,(60.9±5.64)%,(89.12±7.18)%,100,500,1000,10000 ng/ml LPS group compared with the control group,there were significant differences,with statistical significance(P<0.05).2、The HK-2 cells were pretreated with different concentrations of HBSP for 2 h,followed by 500ng/ml LPS action for 24h.The cell viability and apoptosis were examined.Cell activity(fig 2A)in each group was(83.5 ± 1)%,(51.5 ± 1.4)%,(56.9 ± 0.6)%,(60.9 ±0.9)%,(73.0 ± 0.9)%,(74.2 ± 1.5)%.Compared with the LPS group,the cell viability of 50,125ng/mlHBSP group increased significantly.The differences were statistically significant(P<0.05).The rate of apoptosis(fig 2B and 2C)was(2.60±)0.42)%,(45.82 ± 5.14)%,(36.53 ±4.61)%,(25.54 ± 3.89)%,(18.97±2.92)%,(17.73 ±1.98)%.Compared with the LPS group,the apoptosis rate of 25,50,125ng/ml HBSP group decreased obviously.There were significant differences,with statistical significance(P<0.05).3、HK-2 is divided into negative control group,HBSP group,LPS group,HBSP+LPS group,and ROS(fig 2D)of each group was(15.75± 0.29)%,(9.94 ± 0.38)%,(105.88 ±6.17)%,(31.58± 1.18)%.Compared with LPS group,the ROS of HBSP+LPS group decreased significantly.There were significant differences,with statistical significance(P<0.05).4、HK-2 is divided into negative control group,HBSP group,LPS group,HBSP+LPS group,and MDA(fig 2E)of each group was(4.62± 0.36)%,(4.05± 0.24)%,(13.96±0.59)%,(7.14±0.50)%.Compared with LPS group,the MDA of HBSP+LPS group decreased significantly.There were significant differences with statistical significance(P<0.05).Research conclusion1、LPS can reduce the vitality of renal tubular epithelial cells in a dose-dependent anner,Increase apoptosis,and cell activity increased and apoptosis decreased after HBSP added.2、LPS can lead to significant increase of ROS and MDA in renal tubular epithelial cells,causing oxidative stress injury,while ROS and MDA are significantly reduced after pretreatment of HBSP,and oxidative stress injury is reduced.Part II:The effects of HBSP on PI3K-Akt and Nrf2 pathway in lipopolysaccharide-induced tubular epithelial cellsResearch purposes1、To observe the effect of the PI3K inhibitor LY294002 on the protective effect of HBSP on renal tubular epithelial cells.2、The mechanism of HBSP anti-oxidative stress was discussed from the inherent anti-oxidative pathway PI3K-akt and Nrf2 pathway,which provided a basis for the treatment of renal injury caused by HBSP.Research method1、Cells of human renal tubular epithelial cells(HK-2)cultured in vitro and divided into(1)control group(2)Solvent control group(DMSO group)(3)HBSP group(4)LPS intervention group(5)LPS+HBSP intervention group(6)LPS+ 25 mol/L LY294002 intervention group(7)LPS+HBSP+ 25 mol/L LY294002 intervention group.MTT assay was used to measure cell viability,Annexin V-FITC/PI staining combined with flow cytometry to detect the apoptosis of renal tubular epithelial cells.Western-blotting was used to detect the expression of total AKT,P-AKT,Nrf2,HO-1,and NQO1 in the blank control group,the HBSP intervention group,the LPS intervention group,the LPS+HBSP intervention group,the LPS+25 mol/L LY294002 intervention group and the LPS+HBSP+ 25mol/L LY294002 intervention group.2、Cells of human renal tubular epithelial cells(HK-2)were divided into(1)negative control group(2)consiRNA group(3)Nrf2 siRNA group(4)LPS + HBSP group(5)LPS + HBSP+Nrf2 CON group siRNA(6)LPS + HBSP+Nrf2 siRNA group,the cells in the logarithmic growth phase were inoculated in serum-free medium of the 6 well plate.According to the operation procedure provided by lipofectamine 2000,the final concentration of si RNA was 50 nmol/L,and then the complete medium was used for the next step.Western-blotting method was used to detect the expression of HO-1 and NQO1 of each group.Research results1、There was no significant difference in the survival rate and apoptosis rate of renal tubular epithelial cells between the DMSO group and the blank control group,and the effect of DMSO on the cell damage could be excluded in the experiment.The survival rate of renal tubular epithelial cells in LPS group was significantly than that in the control group,and the cell apoptosis rate increased significantly,and the difference was statistically significant(P<0.05).The survival rate of renal tubular epithelial cells in group HBSP+LPS,was significantly higher than that in group LPS,and the apoptosis rate decreased significantly.In the HBSP+LPS group,the PI3K inhibitor LY294002 was given in advance,and the survival rate of renal tubular epithelial cells decreased significantly,and the apoptosis rate was significantly increased.2、The phosphorylation of Akt(p-Akt)in the LPS group was higher than that in the negative control group,and the expression of p-Akt in the HBSP+ LPS groups was significantly increased compared with the LPS group.After pretreatment with PI3K inhibitor HK-2,the expression of p-Akt in the LY294002 group was significantly lower than that in the HBSP + LPS group.3、In the LPS group,HBSP intervention was given in advance,with the decrease of Nrf2 in the cytoplasm and the apparent increase of Nrf2 in the cell nucleus,significantly compared with the LPS group,and the effect wassignificantlyreduced after pretreatment with LY294002.4、Compared with negative control group the expression of antioxidant protein HO-1 and NQO1 increased.The levels of HO-1 and NQO1 expression was significantly increased after treated with HBSP,HO-1 and NQO1 protein expression level decreased significantly after pretreatment with LY294002.5、Nrf2 is an important upstream regulator of HO-1 and NQO1,using Nrf2 siRNA to silence Nrf2 in cells.Compared with LPS + HBSP+Nrf2 con siRNA group and LPS + HBSP group,the expression of Ho-1 and NQO1 in LPS + HBSP+Nrf2 siRNA group decreased significantly.Research conclusion1、The PI3K pathway inhibitor LY294002 can significantly reverse the cellular protective effect of HBSP.2、HBSP can induce Akt phosphorylation and activate the PI3K/Akt pathway.3、The PI3K/Akt signaling pathway mediates HBSP induced nuclear translocation of Nrf2.4、The PI3K/Akt signaling pathway is involved in the induction of HO-1 and NQO1 expression by HBSP.5、The expression of HO-1 and NQO1 induced by HBSP is regulated by Nrf2.6、HBSP through the activation of PI3K/Nrf2 signaling pathway,promoting the expression of downstream antioxidant protein HO-1 and NQO1,thereby increasing the generation of endogenous antioxidant ability,alleviate oxidative stress,reduce sepsis renal oxidative stress and apoptosis.Part III:The protective effect of HBSP.on lipopolysaccharide-induced renal injury in ratsResearch purposesTo investigate the protective effect of erythropoietin-derived peptide(HBSP)on kidney injuries and the possible mechanisms in rats with kidney injury induced by lipopolysaccharide(LPS).Research methods1、Thirty SD rats were randomly divided into 3 groups:blank control group,model group(LPS group)and HBSP drug protection group(LPS+HBSP group),10 rats in each group.Rats in LPS and LPS + HBSP groups were injected with 10 mg/kg LPS intravenously.Rats in the blank control group were injected with an equal volume of saline.30 minutes after LPS injection,HBSP 60ug/Kg was administered to the HBSP-protected group,and the blank group and LPS group were given a corresponding amount of physiological saline as a control.2、After 24 hours of LPS injection,the rats were sacrificed by ether anesthesia.Serum BUN and creatinine(Cr)levels were measured by automatic biochemical analyzer.Serum TNF-α levels were measured by ELISA.The serum and renal homogenate MDA of rats were measured by thiobarbituric acid method.3、The right kidney was cut,part of the kidney was fixed with 10%formaldehyde,alcohol was dried,paraffin was embedded and sliced,and the apoptosis of renal tubular epithelial cells was detected by TUNEL method.4、The left kidney was cut and frozen in-80 refrigerator.The total protein and nucleocapsid protein were extracted.The expression of Nrf2,HO-1,NQO1,AKT and p-AKT in the kidneys of rats were detected with Westerb blot.Research results1、Compared with the control group,the serum urea nitrogen and creatinine in the LPS group increased significantly(P<0.05).The serum urea nitrogen and creatinine in the LPS+HBSP group were significantly higher than those in the control group(P<0.05),but significantly lower than those in the LPS group(P<0.05).Compared with the control group,the activity of TNF-alpha was significantly increased in group LPS and group LPS+HBSP(P<0.05),and the activity of TNF-alpha in LPS +HBSP group was lower than that in LPS group(P<0.05).The results are shown in Table 1.2、Compared with the control group,serum MDA and MDA in the kidney tissue of the LPS group and the LPS+HBSP group were significantly higher(P<0.05).Compared with the LPS group,serum MDA and significant MDA in the kidney tissue of the LPS+HBSP group decreased(P<0.05).The results are shown in Table 2.3、The apoptosis of renal tubular epithelial cells was significantly increased in LPS group and LPS+HBSP group compared with control group(P<0.05).Compared with LPS group,the apoptosis of renal tubular epithelial cells was significantly decreased in LPS+HBSP group(P<0.05).The result is shown in Figure 1.4、There was no significant difference in the expression of Nrf2,HO-1,and NQO1 protein in kidney tissue between the control group and the LPS group.Compared with the control group and the LPS group,the expression of Nrf2,HO-1,and NQO1 protein in the renal tissue of the LPS+HBSP group was significantly increased(P<0.05).The results are shown in Figure 2A and Figure 2B.5、There was no significant difference in the expression of AKT protein in the kidney of the three groups.There was no significant difference in the expression of p-AKT protein between the control group and the LPS group.Compared with control group and LPS group,the expression of p-AKT protein in kidney tissue of LPS+HBSP group was significantly increased(P<0.05).The result is shown in Figure 2C.Research conclusion1、HBSP can reduce the renal injury of LPS rats by increasing the expression of Nrf2 and promoting its nuclear translocation to activate the expression of downstream antioxidant genes HO-1 and NQO1.2、PI3K/AKT may mediate the renal protective effect of HBSP.3、HBSP is expected to become a new treatment strategy for acute kidney injury in sepsis.