Effects And Mechanisms Of Rutaecarpine Derivative Ru-4 On Nephrotoxic-induced Acute Kidney Injury

Background and objective: Acute kidney injury(AKI)is a clinical syndrome that is caused by an Acute inflammatory response and characterized by a rapid decline in renal function,leading to severe kidney damage.Causes of AKI include drug toxicity,ischemia reperfusion,sepsis,etc.AKI is one of the most common complications of hospitalized patients,and its incidence is increasing,especially in the elderly with high morbidity and mortality.Although a certain proportion of patients with AKI have kidney self-repair process,severe or recurrent AKI may still turn into chronic kidney disease(CKD)or even end-stage kidney disease.Because the current treatment of AKI only includes symptomatic support therapy and renal replacement therapy,there is still a lack of effective therapeutic drugs and key targets,so it is of great significance to find therapeutic drugs for AKI.Traditional Chinese medicine(TCMs)refers to natural medicines and their processing substitutes,mainly including plant medicines,animal medicines and mineral medicines,which are guided by the theories of TCMs and have a unique theoretical system and application form,and are used to prevent and treat diseases and have the function of rehabilitation and health care.Due to its low toxicity and side effects,more and more studies have attempted to apply TCMs in the treatment of clinical diseases in recent years,and the successful application of artemisinin in the treatment of malaria has confirmed the potential value of natural compounds.Through the establishment of AKI models in vitro and in vivo,our research group preliminarily identified a series of CMs monomers and their derivatives with anti-inflammatory effects,including Aloin,Hanbaicalein,Puerarin,Icariin,Protocatechuic acid,Protocatechualdehyde and Rutaecarpine.The results of previous studies showed that the chemical monomer Rutaecarpine(Ru)extracted from TCMs evodipine had a good protective effect on the injury of renal tubular epithelial cells.We obtained a series of 3-aromatic sulfonamide-substituted derivatives of Rutaecarpine by modifying its structure,increasing its selectivity and reducing its side effects.This study further explored the protective function and mechanism of Rutaecarpine derivatives on cisplatin-induced acute kidney injury,and sought for potential therapeutic targets and new ideas for AKI.Methods: In vitro study: Human tubular epithelial cells(HK2)from the 6th to 15 th generation were selected for the experiment.First,the optimal therapeutic concentration of the drug was screened by MTT.The experiment was divided into six groups: normal control group,single dosing group,cisplatin model group,low dose dosing group,medium dose dosing group and high dose dosing group.Protein and m RNA were extracted and the expression of kidney injury factor 1(KIM-1)was detected by Western blot,Immunofluorescence and Real-time PCR.Western blot and Real-time PCR were used to detect the expression of renal inflammatory factor TNF-?,IL-1?,IL-6,and the phosphorylation of key inflammatory pathway protein NF-kB-P65.Western blot was used to detect the expression of RIPK1/RIPK3/MLKL,key proteins of programmed necrosis and the changes of Cleaved-Caspase3,the gold marker of apoptosis.Cell apoptosis was detected by Flow cytometry.Finally,Western blot was used to detect the expression of NOX1,NOX2 and NOX4,important members of the NOX family of oxidative stress,and DCF and DHE were used to detect the level of intracellular oxidative stress.The public prediction website DS2017 was used to predict drug targets,and the combination of drug and target PDE4 B was verified by molecular docking and CESTA experiments.The expression of PDE4 B was detected by Western blot and Immunofluorescence.The expression of PDE4 B was silenced by si RNA transfection.Western blot and Real-time PCR were used to detect the effect of Ru-4 on inflammatory response and programmed cell death of renal tubular epithelial cells after PDE4 B silencing.The PDE4 inhibitor Rolipram was used as the positive control group,and Western blot and Real-time PCR were used to detect whether the selective inhibition of PDE4 B had a better protective effect.In vivo study: We constructed an acute kidney injury model by intraperitoneal injection of cisplatin(20 mg/kg)into mice,and treated them with Ru-4(25,50,and 100 mg/kg).The experiment was divided into six groups(n=8): normal control group,single administration group,cisplatin model group,low dose administration group,medium dose administration group,and high dose administration group.After successful modeling,the renal function of the mice in each group was observed by blood creatinine and urea nitrogen test,and the morphological changes of renal tubules in each group were compared by PAS staining.The expression of kidney injury factor 1(KIM-1)was detected by Western blot,immunohistochemistry and real-time PCR.Western blot,Immunohistochemistry,and Real-time PCR were also used to detect the release of renal inflammatory cytokines TNF-?,IL-1?,and IL-6,the phosphorylation of key inflammatory pathway protein NF-kB-P65,and the expression changes of drug target protein PDE4 B.Finally,Western blot was used to detect the expression of RIPK1/RIPK3/MLKL,the changes of Cleaved Caspase3,the gold indicator of apoptosis,and the expression of NOX1,NOX2 and NOX4,important members of the NOX family of oxidative stress.Results In vitro study: MTT results showed that Ru-4 and Ru-7 had the best cellular protective effects among the derivatives of Rutaecarpine.Meanwhile,Western blot results showed that Ru-4 was better than Rutaecarpine and Ru-7 in reducing the damage of renal tubular epithelial cells.Immunofluorescence and Real-time PCR also confirmed that Ru-4 could reduce the expression of KIM-1.Western blot and Real-time PCR also showed that Ru-4 could reduce the release of inflammatory cytokines TNF-?,IL-1?,and IL-6,and the phosphorylation of NF-kB-P65.In addition,Flow cytometry results showed that the level of apoptosis was significantly reduced after Ru-4 treatment,and Western blot also showed that the key proteins of programmed necrosis,RIPK1/RIPK3/MLKL,and the golden marker of apoptosis,Cleaved-Caspase3,were decreased.Finally,DCF staining,DHE staining and Western blot also confirmed that ru-4 can reduce the occurrence of oxidative stress.Phosphodiesterase 4B(PDE4B)was confirmed as a possible target of Ru-4 by molecular biological prediction,molecular docking and cell thermal displacement experiments(CESTA).Western blot and Immunofluorescence also showed that Ru-4 could reduce the expression of PDE4 B protein and increase the content of c AMP.At the same time,after Ru-4 treatment was performed on the silenced PDE4 B cells,Western blot results showed that PDE4 B silencing could reduce the damage of renal tubular epithelial cells,but the drug could not further reverse the cisplatin induced cell damage.At the same time,Western blot also showed that after adding the PDE4 inhibitor rolipram,the targeted inhibition of PDE4 B had a better protective effect in cisplatin treated renal tubular epithelial cells.So we show that Ru-4 works through a PDE4B-dependent pathway.In vivo study:Serum creatinine and urea nitrogen showed that Ru-4 could significantly reverse cisplatin-induced renal function decline.Similarly,renal PAS staining showed that the drug alleviated renal tubules dilatation and glycogen deposition.Western blot,Immunohistochemistry and Real-time PCR confirmed that acute kidney injury marker KIM-1 was also reduced.We also found that Ru-4 can alleviate cisplatin-induced programmed cell death,inflammatory response and oxidative stress in the kidney.Conclusion(1)In vitro study showed that Ru-4 inhibited cisplatin-induced renal tubular epithelial cell injury,inflammatory response,programmed death and oxidative stress.(2)In vitro study showed that Ru-4 could not further reduce cisplatin-induced HK2 cell injury,inflammatory response and programmed death in PDE4B-silenced cells,suggesting that Ru-4 may have a protective effect on cisplatin-induced HK2 cell injury through a PDE4B-dependent mechanism.(3)In vivo study showed that Ru-4 effectively inhibited renal function decline,programmed cell death,inflammatory response and oxidative stress caused by cisplatin induced AKI in mice.