Safety And Efficacy Of Twist1 Inhibitor Harmine In Alleviating Renal Fibrosis

Background:At present,about 850 million people worldwide suffer from different types of kidney diseases,and up to 10%of adults suffer from chronic kidney disease（CKD）.Renal fibrosis is the common pathological manifestation of various CKD progression.At present,there are only drugs that indirectly delay the progression of kidney disease,and there are no effective drugs that directly inhibit fibrosis.Therefore,the study of anti-fibrosis drugs is a crucial step in slowing down the further deterioration of the condition of CKD patients and improving their prognosis and quality of life.Through a series of experiments in vitro and in vivo,our group confirmed that the transcription factor Twist1 plays an important role in promoting renal fibrosis.It is suggested that intervention in Twist1 may become an effective target for anti-fibrosis.Harmine is a kind ofβ-carboline alkaloid that can be found in many plants.In an unbiased screening,Harmine was found to be able to effectively inhibit Twist1,and has significant anti-tumor activity in lung cancer.Harmine treatment can lead to the degradation of Twist1 in NSCLC cells and induce cell aging or apoptosis.However,the role of Harmine as a Twist1 inhibitor in renal fibrosis has not been reported.To this end,we explored the safeness of Hamrine in vivo and in vitro through MTT test and LD₅₀test,and verified the effect of Harmine on inhibiting renal fibrosis by inhibiting Twist1 in vivo and in vitro,laying a foundation for further exploring the preclinical study of Harmine’s anti-fibrosis effect.Aims:The purpose of this study is to explore the effectiveness and safeness of the Twist1 inhibitor Harmine in inhibiting renal fibrosis and lay a foundation for further exploration.1.Observe the expression of Twist1 in tissues of patients with different types of kidney disease and analyse the relevance of Twist1 and fibrosis and the e GFR of patients.2.Preliminary safeness evaluation of Harmine in vivo and in vitro.3.Research on the effectiveness of Harmine in the treatment of renal fibrosis.Methods:1.Collect kidney biopsy tissue samples from patients with various chronic kidney diseases,and carry out immunohistochemical staining to observe the correlation between Twist1 and renal fibrosis and e GFR.2.MTT experiments were carried out with human renal tubular epithelial cell lines HK-2,HKC-8,mouse renal tubular epithelial cell line BUMPT and rat renal tubular epithelial cell line NRK-52E respectively.The IC₅₀of Harmine in four kinds of cells was calculated for 24 hours,48 hours and 72 hours respectively,so as to evaluate the safeness of Harmine in vitro.With different concentration gradients,C57BL/6J mice were injected intraperitoneally with Harmine for LD₅₀detection.The reaction and status of mice within60 minutes after administration were observed.The body weight was recorded before administration every day.One week later,important organs were taken for HE staining and serum liver and kidney function test to evaluate the safeness of Harmine in vivo.3.The hypoxia incubator was used to treat HK-2 cells in two time gradients of 24 hours and 48 hours,and the administration of Harmine was conducted at the same time.Three appropriate concentration gradients were set according to the previous LD₅₀results,and the inhibition of Harmine on Twist1 and fibrosis were evaluated at the protein and RNA levels.The model of unilateral renal ischemia-reperfusion（u IRI）renal fibrosis was established by using WT,Wild Type C57BL/6J mice and Twist1^{c KO}mice.The mice were injected intraperitoneally with different concentration gradients of Harmine solution.At the same time,a group of u IRI+benazepril hydrochloride tablets was established as a positive control group.After 14 days,the kidney tissue was taken for HE,Masson,PAS staining and immunohistochemistry（IHC）staining,and the effectiveness of Harmine in inhibiting renal fibrosis was evaluated at the protein and transcription levels.Results:1.Observe the renal biopsy tissue samples of patients with different types of chronic kidney disease,and find that the expression of Twist1 is positively correlated with the degree of fibrosis,and the expression of Twist1 is higher in the kidney tissue with more fibrosis.However,with the elevation of Twist1 expression,the e GFR of patients descends.2.The IC₅₀values of four cell lines at 24 h,48 h and 72 h were successfully calculated by MTT experiment.In HK-2 cells,the IC₅₀values of Harmine at 24 h,48 h and 72 h were86.44 respectivelyμmol/L,89.18μmol/L,and 30.50μmol/L;In HKC-8 cells,the IC₅₀ values of Harmine at 24 h,48 h and 72 h were 117.61μmol/L,20.72μmol/L,and 18.30μmol/L respectively;In BUMPT of mouse renal tubular epithelial cells,the IC50 values at three time points were 21.82 respectivelyμmol/L,11.32μmol/L,and 6.31μmol/L;In NRK-52E cells,the IC₅₀values of Harmine at 24 h,48 h and 72 h were 51.43μmol/L,19.82μmol/L,and 16.16μmol/L respectively.It provides a concentration reference for the subsequent cell dosing treatment experiment,and evaluates the safeness of Harmine in vitro.It was successfully calculated that the LD₅₀of Harmine in C57BL/6J mice was36.87 mg/kg.In addition,according to the behavior of mice after administration,daily body weight changes and serum liver and kidney functions,it was found that when the concentration of Harmine administered intraperitoneally exceeded 20 mg/kg,mice began to increase their activity and hyperactivity.With the further increase of the concentration,mice developed convulsions,tremors,ataxia and even poisoning death.However,no difference was found in the histopathological staining of heart,liver,spleen,lung and kidney and the detection of serum liver and kidney function.3.When Harmine was added to HK-2 cell culture medium and hypoxia treatment was conducted for 24 h and 48 h,the expression of Twist1 and fibrosis index was down-regulated in a time-dependent and dose-dependent manner.Intraperitoneal injection of Harmine into u IRI mice could inhibit the expression of Twist1 and alleviate the fibrosis phenotype of u IRI mice.The phenotype of fibrosis reduction was consistent with that of Twist1^{c KO}mice and u IRI+benazepril hydrochloride positive control group.Conclusion:1.The expression of Twist1 was positively correlated with the degree of fibrosis in the tissue samples of chronic kidney disease of different pathological types while negatively correlated with e GFR of patients.2.The IC₅₀of Harmine in different cell lines is different.The LD₅₀value of Harmine in mice is 36.87 mg/kg.After intraperitoneal injection of Harmine more than 20 mg/kg,the mice begin to have nervous excitation symptoms,and when the concentration is too high,the symptoms of neurotoxicity will occur.However,no significant difference was found in the detection of liver and kidney function in serology and histopathological staining of important organs.3.The expression of Twist1 in human renal tubular epithelial cells induced by hypoxia and the fibrosis phenotype can be inhibited by Harmine in a time-dependent and dose-dependent manner.In the renal fibrosis model induced by u IRI model,intraperitoneal injection of Harmine can alleviate the renal fibrosis phenotype.The phenotype was consistent with that of Twist1^{c KO}mice and benazepril hydrochloride positive control group.