Study On The Protective Effect And Mechanism Of Dihydroartemisinin On Acute Renal Injury

Objective:Sepsis refers to systemic inflammatory response syndrome(SIRS)caused by infection.Sepsis is a fatality disease with a high mortality rate,and about 14,000 people die every day from complications of sepsis in the world.According to foreign epidemiological survey,sepsis has been the main cause of death in intensive care unit(ICU)patients,the mortality of sepsis has exceeded myocardial infarction,and has become the main cause of death of non-cardiac patients in ICU.Sepsis causes acute kidney injury(AKI),which accounts for 15% to 20% of all ICU patients,and the probability of death caused by sepsis is as high as 60%.The pathophysiology of AKI caused by sepsis includes severe renal parenchymal inflammation,endothelial dysfunction,glomerular thrombosis,and renal tubular injury.Vascular endothelial dysfunction plays a key role in the microcirculation disorder of sepsis.When endothelial cells are exposed to a variety of inflammatory mediators,endothelial cell structure changes,cell connections are lost,and the destruction of polysaccharide-protein complexes leads to an increase in endothelial permeability.Glomerular endothelial cell damage and the increase of the permeability will eventually lead to damage of glomerular filtration barrier(GFB).The damage of GFB is the most important clinical feature of AKI,preventing the injury of GFB is significant to the treatment of sepsis induced AKI.Artemisinin is a compound of sesquiterpene lactone,which is extracted from the stem leaves of the composite inflorescence plant by Chinese scientist Tu youyou in 1971.Dihydroartemisinin(DHA)is a semi-synthetic derivative of artemisinin,with less side effect and more efficient.In addition to treat malaria effectively,artemisinin has also been found to exhibit anti-tumor,anti-inflammatory and anti-angiogenic effects.Artemisinin can effectively reduce the symptoms of lupus nephritis in mice.In addition,the artemisinin derivative 905 inhibits collagen-induced arthritis.These findings suggest that artemisinin-based drugs have the potential to treat inflammation-related diseases.In this study,we will focus on the protective effects of DHA in sepsis induced AKI.by constructing an(LPS)induced sepsis model in mice and in vitro cell experiments,we will explore the inhibitory effect and potential mechanism of DHA treatment on inflammatory mediated increase in permeability of renal endothelial cells.Research Methods:1 To construct the mouse model of sepsis,6-8 weeks old C57BL/6 mice were used and intraperitoneal injection of LPS(20mg/kg)was performed to induce the mouse model of sepsis.The mice were randomLy divided into four groups,containing control group and DHA treatment group injected phosphate buffer saline(PBS),other two groups were injected with LPS to form the model of sepsis.After an hour of intraperitoneal injection,the model mice were administrated DHA(50mg/kg)by means of intragastric administration contrasting with phosphate buffer saline(PBS)to compare the effect.2 To detect the effect of DHA treatment on the kidney of mouse model,the urine and blood were collected after 24 hours of intragastric administration.The ratio of urine albumin/urinary creatinine and the concentration of blood creatinine were detected.3 To detect the pathological changes of kidney in the mouse model of sepsis,the mice were anesthetized and kidney was separated.HE staining in frozen sections of mouse kidney was used to detect the pathological changes of kidney of mice.4 In vitro experiments,20 ng/mL TNF-α was used to culture human renal glomerular endothelial cells(HRGECs)for 24 hours to simulate inflammation.HRGECs were treated with DHA(25 μM)、TNF-α and DHA combined with TNF-α for 24 hours.And then,fluorescein isothiocyanate-glucan transwell experiment and ECIS experiments were used to detect the effect of DHA on the increase of cell permeability induced by TNF-α.5 To test the effect of DHA on viability of HRGECs,25 μM DHA was used to culture HRGECs alone for 24 hours.Flow cytometry and trypan blue staining were used to assess the effects of DHA on apoptosis and cell viability of HRGECs.6 By using immunofluorescence staining and Western blot,the effect of DHA on occludin expression in HRGECs treated by DHA(25 μm)、TNF-α and DHA combined with TNF-α was detected.Results:1 The mouse model of sepsis resulted in AKI in mice: after intraperitoneal injection with LPS for 24 hours,the results of UACR and serum creatinine values test showed that LPS increased UACR and serum creatinine values when compared with control group(intraperitoneal injection with PBS).HE staining showed that intraperitoneal injection with LPS induced glomerular hemorrhage,edema and infiltration of inflammatory cells in the kidney of AKI mice.2 DHA significantly reduced the increase of UACR and serum creatinine values induced by LPS injection in sepsis mice: the results of UACR showed that DHA treatment alone did not affect UACR compared with control group.However,DHA treatment after LPS injection significantly reduced UACR compared with LPS injection group.Serum creatinine test showed the same result,DHA treatment after LPS injection significantly reduced serum creatinine values compared with LPS injection group.3 DHA attenuated the injure of AKI induced by LPS injection in sepsis mice: HE staining showed that DHA treatment significantly reduced the glomerular hemorrhage,edema and infiltration of inflammatory cells after intraperitoneal injection with LPS in the kidney of AKI mice.DHA treatment maintained glomerular integrity.4 DHA treatment reduced the permeability of HRGECs which was elevated by TNF-α.ECIS test showed that TNF-α treatment reduced the resistance of HRGECs.When HRGECs were cultured with DHA and TNF-α,the resistance value was significantly increased which suggested that the permeability was reduced.At the same time,we showed that DHA alone did not affect the resistance value of HRGECs.Transwell permeability experiment of isothiocyanate found the similar results,that DHA and TNF-α co-treatment decreased the rate of cell transition compared with TNF-α group.5 DHA treatment had no effect on cell apoptosis and viability of HRGECs: flow cytometry showed that DNA treatment alone did not induced apoptosis of HRGECs compared with control group(treated with PBS).Trypan blue staining showed that There was no significant change in the percentage of living cells in DHA treatment group compared with the control group.6 TNF-α reduced the expression of occludin in HRGECs,and DHA co-treatment inhibits this affect.Both immunofluorescence and Weatern blot showed that the protein level of occludin in HRGECs was reduced by TNF-α,however,DHA combined with TNF-α significantly inhibited the reduction of occludin.Conclusion:1 In AKI model of sepsis induced by LPS,oral DHA decreases the increase of UACR induced by LPS;oral DHA also relieves glomerular hemorrhage,edema and infiltration of inflammatory cells in the kidney of AKI mice induced by LPS,while taking DHA orally has no effect on the kidney of normal mice.The results suggest that DHA itself did not damage the kidney of mice and had therapeutic effect on AKI.2 DHA does not affect the activity of HRGECs and induce HRGECs apoptosis,indicating that DHA itself has no toxic effect on HRGECs.3 TNF-α treatment increases the permeability of HRGECs.DHA combined with TNF-α inhibits the increase of HRGECs permeability caused by TNF-α,which indicates that DHA could inhibit the increase of HRGECs permeability induced by TNF-α under inflammatory conditions.4 TNF-α treatment decreases the expression of occludin in HRGECs.DHA and TNF-α co-treated restore the expression of occludin in HRGECs.DHA maintained the expression of occludin in inflammatory condition,suggests that DHA may maintain the permeability of endothelial cells by maintaining the expression of tight junction protein occluding.Significance:Sepsis is a systemic inflammatory response syndrome.The treatment is difficult and expensive,and it can bring physical and mental pain to patients while consuming huge medical resources,which can seriously affect the quality of life of patients and even threaten their lives.AKI is the most common and serious complication of sepsis.At present,the treatment of AKI is mainly based on renal replacement therapy(RRT),and there is no reliable and effective drug to treat AKI.Artemisinin is a widely used antimalarial,and artemisinin with its derivatives also exhibit anti-tumor,anti-angiogenic and anti-inflammatory effects.Artemisinin can effectively relieve the symptoms of lupus nephritis in mice.Our results showed that oral DHA relieved glomerular hemorrhage,edema and infiltration of inflammatory cells in the kidney of AKI mice induced by LPS.In vitro experiment also showed that by maintaining expression of occluding,DHA inhibited the increase of permeability of glomerular endothelial cells induced by TNF-α.This suggests that DHA may have a therapeutic effect on sepsis induced AKI.This provides experimental support and theoretical basis for the future safety application of DHA in treating sepsis induced AKI.