Study On The Anti-type 2 Diabetes Effect Of Celastrol And Its Underlying Mechanism

BackgroundDiabetes mellitus(DM)is a systemic chronic metabolic disease characterized by hyperglycemia.Long-term metabolic disorders lead to many complications,such as coronary heart disease,stroke,retinopathy,and diabetic nephropathy,and have a long course of the disease,poor prognosis,and high mortality.According to the International Diabetes Federation(IDF),more than 537 million people have been diagnosed with DM worldwide in 2021,and about 5 million people died due to DM.Type 2 diabetes mellitus(T2DM)is the most common type of diabetes,accounting for 90-95%of cases,and has become the third-largest chronic disease after cancer and cardiovascular disease.Although pharmacological intervention remains the primary line of treatment for T2DM,the existing therapeutic drugs have certain limitations in clinical treatment.Thus,it is always the hotspot to develop safe and effective hypoglycemic drugs with new targets.In addition,most patients with T2DM are elderly,and suffer from multiple underlying diseases at the same time,such as obesity,cancer,cardiovascular diseases,etc.,As a result,they need to take multiple drugs at the same time for a long time,which not only exposes the patients to potential risks of many drug side effects,but also brings a heavy burden to individuals and society.The research and development of drugs with multiple pharmacological activities at the same time and the elucidation of their corresponding pharmacological mechanisms have important scientific significance and significant social benefits.Celastrol(Cel)has a very wide range of pharmacological effects,including anti-rheumatism,anti-tumor,anti-Alzheimer’s disease,etc.,and has been listed as one of the 5 traditional natural medicinal compounds which are most likely to be developed into modern medicine by the Cell(2007,Vol.130,No.5).Studies have shown that celastrol can reduce insulin resistance,improve insulin sensitivity,and reduce kidney and liver damageand decrease the body weight in db/db diabetic mice.Although existing studies have shown that celastrol has the potential in treatment of diabetes and its complications,its hypoglycemic target and specific molecular mechanisms are still not well understood.It is a big obstacle for developing celastrol as a hypoglycemic drug.Therefore,elucidating the hypoglycemic target and molecular mechanism of celastrol has important scientific significance and clinical value.Methods1.The anti-diabetic effects of celastrol(1)db/db mice were used as an animal model of T2DM,the effects of celastrol on blood glucose,body weight,food intake and glucose tolerance in vivo were examined;the effect of celastrol on pancreatic insulin secretion was assessed by immunohistochemistry(IHC);(2)A high glucose cell model was established using rat insulinoma INS-1 cells;enzyme-linked immunosorbent assay(ELISA)was used to detect the effect of celastrol on insulin secretion;Western blotting was used to detect the effect of celastrol on the expression of insulin expression-related genes insulin receptor β(IR-β)and musculoaponeurotic fibrosarcoma oncogene family,protein A(MafA);(3)The effect of celastrol on glucose uptake in human hepatoma HepG2 cells was detected by 2-NBDG assay;the effect of celastrol on the expression of gluconeogenesis-related genes phosphoenolpyruvate carboxy kinase 1(PCK1)and glucose-6-phosphate dehydrogenase deficiency(G6PD)was examined by western blotting.2.Effect of celastrol on thioredoxin-interacting protein(TXNIP)expression(1)Real-time PCR was used to detect the effects of celastrol on TXNIP mRNA expression in INS-1 and HepG2 cells;western blotting was used to detect the effect of celastrol on TXNIP protein expression in INS-1 and HepG2 cells;IHC was used to detect the effect of celastrol on TXNIP experssion in pancreas and liver of db/db mice.(2)Protein synthesis inhibitor cycloheximide(CHX)was used to block protein synthesis,and CHX chase assy was used to examine the effect of celastrol on TXNIP stability in INS-1 and HepG2 cells.mRNA synthesis inhibitor Actinomycin D(ActD)was used to block transcription,and real-time PCR was used to detect the effects of celastrol on TXNIP mRNA stability after INS-1 and HepG2 cells were treated with ActD(3)Dual-luciferase reporter assay was used to detected the effect of celastrol on TXNIP promoter activity;3.Effect of celastrol on carbohydrate-responsive element-binding protein(ChREBP)expression and nuclear translocation(1)Western blotting was used to detect the effect of celastrol on ChREBP protein expression in INS-1 cells,HepG2 cells and pancreas and liver of db/db mice;(2)The nuclear protein and cytoplasmic protein extraction kit were used to separate cytoplasm and nuclei proteins in INS-1 and HepG2 cells;western blotting was used to detect the effect of celastrol on the expression of ChREBP protein in the cytoplasm and nucleus of INS-1 and HepG2 cells;(3)Immunofluorescence(IF)was used to detect the effects of celastrol on the nuclear translocation of ChREBP in INS-1 and HepG2 cells.4.Mechanisms of celastrol downregulating ChREBP(1)Molecular docking was used to evaluate the binding activity of Cel and ChREBP;Drug affinity responsive target stability(DARTS)experiment combined with mass spectrometry analysis was used to examine the interaction between celastrol and ChREBP;Cellular thermal shift assay(CETSA)was used to detect the intracellular interaction of celastrol and ChREBP;(2)CHX chase assay examined the effect of celastrol on ChREBP protein stability in INS-1 and HepG2 cells;real-time PCR was used to detect the effect of celastrol on ChREBP mRNA stability in INS-1 and HepG2 cells after treatment with ActD;western blotting was used to detected the effect of celastrol on the expression of exogenous ChREBP in HepG2 cells.(3)Western blotting was used to detect the effect of proteasome inhibitor MG 132 and lysosomal inhibitor chloroquine(CQ)on the down-regulation of ChREBP protein levels in INS-1 and HepG2 cells by celastrol;ChREBP was overexpressed in HepG2 cells,and immunoprecipitation(IP)experiments were used to detect the effects of celastrol on degradation of ChREBP by ubiquitination.5.Validation of celastrol ameliorating T2DM via ChREBP/TXNIP axis(1)The plasmid pCDNA3.1-FLAG-ChREBP was constructed and transfected into HepG2 cells.Western blotting was used to detect the effect of celastrol on the protein expression of ChREBP,TXNIP,G6PD and PCK1;real-time PCR was used to detect the effect of celastrol on the mRNA expression of TXNIP.(2)siRNA was used to knock down ChREBP,and western blotting was used to detect the effects of celastrol on insulin expression-related genes IR-β and MafA in INS-1 cells;ELISA was used to detect the effect of celastrol on insulin secretion in INS-1 cells;(3)siRNA was used to knock down ChREBP,and western blotting was used to detect the effects of celastrol on gluconeogenesis-related genes PCK1 and G6PD in HepG2 cells;Results1.Celastrol ameliorates T2DM.(1)Celastrol treatment significantly reduced blood glucose level in db/db mice.After 6 weeks of administration of celastrol at 0.25 mg/kg,0.5 mg/kg,and 1 mg/kg,the hypoglycemic rates were 32.04±4.63%,37.08±6.18%,and 73.73±4.63%,respectively.In addition,celastrol reduced the food intake and body weight,and improved glucose tolerance of db/db mice;(2)Celastrol restored the secretion of insulin in INS-1 cells in the presence of high glucose,and up-regulated the protein expression of insulin expression-related genes IR-β and MafA;(3)Celastrol reversed glucose uptake ability of HepG2 cells,which impaired by high glucose and inhibited the proteins expression of gluconeogenesis-related genes PCK1 and G6PD.2.Celastrol downregulates TXNIP at the transcriptional level.(1)Celastrol treatment reduced TXNIP mRNA and protein levels in both INS-1 and HepG2 cells;celastrol reduced TXNIP levels in the pancreas and liver of db/db mice;(2)Compared with the CHX alone,the CHX combined with celastrol did not further reduce the TXNIP protein levels in INS-1 and HepG2 cells,indicating that celastrol does not affect TXNIP protein stability;Compared with the ActD alone,the ActD combined with celastrol did not further reduce the TXNIP mRNA levels in INS-1 and HepG2 cells,indicating that celastrol does not affect TXNIP mRNA stability.The dual-luciferase enzyme reporter gene results showed that celastrol can inhibit the activity of TXNIP promoter,indicating that celastrol inhibits TXNIP transcription.3.Celastrol binds to ChREBP directly to promote its ubiquitination and degradation.(1)The molecular docking result showed that celastrol and ChREBP have two binding sites with binding energies of-9.4 kcal/mol and-9.3 kcal/mol,respectively;DARTS experiments combined with mass spectrometry analysis showed that ChREBP was indeed the target of celastrol;CETSA experiments showed that celastrol can make the thermal melting curve of ChREBP shifts to the right.The above results together indicated that celastrol can directly bind to ChREBP,and ChREBP was the target gene of celastrol;(2)Celastrol treatment down-regulated the expression of ChREBP protein in INS-1 and HepG2 cellsand in pancreas and liver of db/db mice;(3)Celastrol treatment reduced the levels of ChREBP in both nuclear and cytoplasmic fractions in INS-1 and HepG2 cells;(4)Compared with the CHX alone,the CHX combined with celastrol can further reduce the ChREBP protein levels in INS-1 and HepG2 cells,suggesting that celastrol reduces ChREBP protein stability;Compared with the ActD alone,the ActD combined with celastrol did not further reduce the TXNIP mRNA levels in INS-1 and HepG2 cells,indicating that celastrol does not reduce ChREBP mRNA stability.In addition,celastrol can down-regulate the expression of exogenous ChREBP protein,further supporting that celastrol downregulates ChREBP at post-transcriptional level;(5)The lysosomal inhibitor chloroquine(CQ)did not reverse the down-regulation of ChREBP by celastrol.In contrast,the proteasome inhibitor MG 132 could reverse the down-regulation of ChREBP by celastrol.The IP experiment results further showed that celastrol could promote the ubiquitination of ChREBP.The above results indicated that celastrol down-regulates ChREBP through inducting ubiquitination-mediated degradation.4.By targeting the ChREBP/TXNIP axis,celastrol promotes insulin secretion and suppresses gluconeogenesis,thereby exerting hypoglycemic effects.(1)After overexpression of ChREBP in HepG2 cells,both mRNA and protein levels of TXNIP were simultaneously up-regulated;(2)After knockdown of ChREBP in INS-1 cells,the expression of TXNIP protein was down-regulated,and celastrol could not further down-regulate the expression of TXNIP.ChREBP knockdown also decreased MafA expression and blocked celastrol-induced insulin secretion and IR-β expression in INS-1 cells;(3)After knockdown of ChREBP in HepG2 cells,the expression of TXNIP protein was down-regulated,while celastrol did not further down-regulate the expression of TXNIP,and the down-regulation of gluconeogenesis-related gene G6PD by celastrol was reversed.Conclusions1.Celastrol can promote insulin secretion,improve glucose homeostasis and decrease the blood glucose level in db/db mice;2.Celastrol directly binds to ChREBP,the primary transcriptional factor for upregulating TXNIP.By binding ChREBP,celastrol promotes its ubiquitination and degradation by proteosome,and inhibits its nuclear translocation thus suppressing TXNIP expression;3.By targeting the ChREBP/TXNIP axis,celastrol promotes insulin secretion and inhibit gluconeogenesis,thereby ameliorating T2DM.The above results showed that celastrol has a significant hypoglycemic effects,and the mechanism is that celastrol directly binds to ChREBP,promotes the ubiquitination and degradation of ChREBP and inhibits the nuclear translocation of ChREBP,thereby down-regulating the transcription of TXNIP and breaking the vicious cycle of hyperglycemia deterioration due to TXNIP overexpression.In addition,celastrol can reverse TXNIP-mediated insulin resistance,hepatic glucose metabolism disorder,and pancreatic β-cell dysfunction,thereby exerting hypoglycemic effect;Our study reveals that ChREBP is the hypoglycemic target directly bound by celastrol,which providing new clues and ideas for the development of celastrol as a hypoglycemic drug and further reducing its toxic and side effects through the improvement of druggability.In addition,our study provided theoretical and experimental basis for the formulation of drug regimens for T2DM patients,especially those with various underlying diseases.It has scientific significance and application value for celastrol to become a safe and effective emerging drug that meets the requirements of modern medicine.