Keap1/Nrf2 Signaling Pathway Mediates The Onset Of Type 1 Diabetes And The Therapeutic Effect Of Formononetin Against Type 1 Diabetes

Objective:Diabetes,which is a metabolic disease characterized bychronic hyperglycemia,can seriously damage various organs of the human body and induce a series of complications such as cardiovascular disease,neuropathy,kidney disease and eye disease.Type 1 diabetes mellitus(T1DM),also known as insulin-dependent diabetes mellitus,is a type of diabetes in which the body produces an abnormal autoimmune response that causes damage to islet βcells,leading to a reduction in insulin secretion.With the increasing number of clinical cases reported in recent years,the number of people with T1DM worldwide is now increasing by 128,900 per year,and about 54,040 T1DM patients in China.T1DM mainly occurs in the adolescent population.According to statistics from the International Diabetes Federation in 2019,there are nearly 1.1602 million children and adolescent patients(<20 years old)with T1DM worldwide.Because no enough insulin produced in the human body of the T1DM patients,it easily occurs ketoacidosis in these patients.Therefore,the T1DM patients must inject insulin for treatment for life from the onset.However,due to the poor control of blood sugar in those adolescent patients,symptoms of insulin discomfort frequently occur.In addition,excessive insulin injections can also cause various side effects such as hypoglycemia,dizziness,coma,and even death.Nowadays,because of its rapid increase in incidence,accompanied with low onset age and poor prognosis,T1DM has posed a major threat to public health.Thus,effective treatment of T1DM is urgent.The decrease of insulin secretion caused by islet β cell injury is the pathological mechanism of T1DM.Studies have shown that oxidative stress can induce islet β cell damage,which is one of the main factors involved in the development of T1DM.Kelch-like ECHassociated protein 1(Keapl)/Nuclear factor erythroid 2-related factor 2(Nrf2)is the most important antioxidant signaling pathway in the human body.Nrf2 is the key regulatory factor of the cellular antioxidant system,which can activate many kinds of antioxidant genes transcription,improve the ability of cell anti-oxidation,resist the injury of islet cells caused by oxidative stress,suggesting that Keap1/Nrf2 signaling pathway could be closely related to the occurrence and development of T1DM.However,the role of Keap1/Nrf2 signaling pathway in the regulation of T1DM susceptibility is unclear.Promoting islet β cell regeneration is the key to treating T1DM effectively.Currently,the main therapy for patients with T1DM is insulin injection for life.Although the treatment can rapidly reduce blood glucose,it cannot repair islet cell damage to cure T1DM fundamentally.Therefore,it is important to seek alternative therapies that can protect islet cells for the effective treatment of T1DM.Astragali Radix,which mainly contains saponins and flavonoids,has a long history of clinical application in treating diabetes.Formononetin(FMNT),a kind of isoflavone phytoestrogens,is not only one of the active ingredients in Astragali Radix,but also one of the active ingredients of some common traditional Chinese medicine such as angelica,clover,kudzu root,sophoraflavescens and blood vine.Modern pharmacological research suggests that FMNT has anti-oxidative stress,anti-inflammatory,anti-cancer,and hypoglycemic effects.However,whether FMNT can regulate the Keap1/Nrf2 signaling pathway to treat T1DM has still not been well defined.Accordingly,combined with validated experimental studies,the current study established a T1DM micemodel and an injuried islet β cell line model.On the one hand,this study confirmed the intervention effect of Keap1/Nrf2 signal pathway on T1DM susceptibility;on the other hand,elucidated the molecular mechanism of FMNT in regulating Keap1/Nrf2 signaling pathway to treat T1DM.The results of this study would be helpful to clarify the susceptibility mechanism of T1DM,and might provide support and evidence for the clinical application of FMNT during the treatment of T1DM.Methods:1.Established a T1DM model on C57BL/6J mice,and screened the different susceptibility of the mice to T1DM.A model of T1DM was established by tail vein injection of alloxan(60mg/kg)in C57BL/6J mice.The sensitive and tolerate mice were screened and evaluated by the indexes of fasting blood glucose,plasma insulin,insulin expression in pancreatic tissue and HE staining.2.Detected the expression of Keap1/Nrf2 signaling pathway in the sensitive and tolerate TIDM mice.The expression of Keap1/Nrf2 signaling pathway was detected by Western blot and IHC analysis,respectively,in the sensitive and tolerate mice3.Established an injuried islet β cell(MIN6)model,then detectedthe expression of Keapl/Nrf2 signal pathway and ROS levels.Established an injuried islet β cell(MIN6)model by using alloxan incubation.MTT,EdU and apoptosis assay were used to evaluate the injury degree of MIN6 cells induced by alloxan incubation for 6h.Western blot and Confocal assay were used to detect the nuclear translocation of Nrf2 and also measure the expression of Keap1/Nrf2 signaling pathway.Cytometry and Confocal assay were used to detect the intracellular ROS levels.4.Established a T1DM mouse model and evaluated the therapeutic effect of FMNT against T1DM.A T1DM model was established by tail vein injection of alloxan(60 mg/kg)in C57BL/6J mice.FMNT was administered orally at low,medium and high doses(2.5,5,10 mg/kg),the therapeutic effect of FMNT against T1DM was evaluated by detecting fasting blood glucose,plasma insulin,insulin expression in pancreatic tissue and HE staining.5.Investigated the expression of Keapl/Nrf2 signaling pathway in pancreatic tissue of T1DM mice.Detected the expression of Keap1/Nrf2 signal pathway in mice pancreatic tissues of normal control group,model group and FMNT treatment group by Western blot and IHC method,respectively.6.Established an injury MIN6 cell model to investigate the effect of FMNT on relieving alloxan-induced cell injury.Detected the expression of Keap1/Nrf2 signaling pathway and the content of ROS levels in MIN6 cells.3 mM alloxan was used to established an injury MIN6 cells.The efficacy of FMNT was evaluated by MTT,EdU,and apoptosis assay,respectively.Western blot and Confocal assay were used to detect nuclear translocation of Nrf2 and measure the expression of Keap1/Nrf2 signaling pathway in cells.And the flow cytometry and confocal assay were also used to detect the expression of ROS levels in cells.Results:(1)There were individual differences in the incidence of alloxan-induced T1DM in C57BL/6J mice.In the T1DM sensitive mice,the weight and plasma insulin were significantly reduced,fasting glucose and liver index and kidney index were significantly increased,with no clear boundary and irregular shape;in the alloxan-induced MIN6 injured cells,the cell viability and proliferation of MIN6 injured cells were decreased significantly,besides,the cell apoptosis and intracellular ROS levels were increased significantly.(2)The Keap1/Nrf2 signaling pathway was significantly inhibited both in the mice that were sensitive to T1DM,and in the MIN6 injured cells.The Keapl protein expression was markedly decreased,the protein levels of Nrf2,HO-1 and NQO-1 were markedly increased.(3)FMNT could significantly reduce the blood glucose of T1DM mice,increase the plasma insulin,protect the β cells from alloxan-induced damage,and produce effective therapeutic effect against T1DM;FMNT could also increase the viability and proliferation of MIN6 injured cells,but reduce the apoptosis and intracellular ROS levels.(4)FMNT could significantly inhibit the protein expression of Keapl,and up-regulate the protein expression of Nrf2 and HO-1 in the T1DM mice.FMNT could also significantly activate the Keap1/Nrf2 signaling pathway,and up-regulate the protein levels of Nrf2,HO1 and NQO-1 to reduce the ROS levels in injured MIN6 cells.Conclusion:On the one hand,Keap1/Nrf2 signaling pathway mediates the onset of T1DM;on the other hand,FMNT can effectively improve T1DM by activating Keap1/Nrf2 signaling pathway.The results of this study would be helpful to clarify the susceptibility mechanism of T1DM,and might provide support and evidence for the application of FMNT during the clinical treatment of T1DM.