Mechanism Of Clock Gene Involved In Diabetic Kidney Injury And Intervention Effect Of Melatonin

Introduction: Along with economic development and lifestyle changes,the risk of diabetes is increasing,and it poses a serious threat to people’s health.Diabetic kidney disease(DKD)is a common complication of diabetes.Its pathogenesis is mainly related to abnormal glucose metabolism,renal hemodynamic changes,immune and inflammatory factors,genetic factors,oxidative stress and so on.A large number of studies show that in the pathogenesis of diabetes,circadian rhythm disorder is one of the causes that can not be ignored.Circadian rhythm is a natural phenomenon in organisms,which is mainly regulated based on the biological clock to make the body adapt to the changes of the natural environment.Relevant studies have found that the biological clock is synchronized with the changes of solar photoperiod and material metabolism.Blood glucose plays an important role in the process of substance and energy metabolism.Relevant experimental studies have found that blood glucose level is closely related to the biological clock gene expression.Abnormal clock genes have an impact on insulin secretion and rhythm.Relevant experimental studies have shown that for DM mice,in the case of circadian rhythm disorder,the blood sugar level is significantly increased,and the renal weight coefficient and serum creatinine index have increased.associated with an increased incidence of DKD.Toll like receptors(TLRs)are membrane receptors,which are closely related to immune regulation.They can trigger inflammatory response through specific molecular patterns,and play an important role in the activation of immune system.Stimulated by hyperglycemia and hyperlipemia,Toll like receptors in podocyte and glomerular mesangial cells are activated,thus promoting the expression and release of cytokines and chemokines,leading to inflammation and fibrosis,and forming pathological changes of diabetic nephropathy.The researchers found that the expression of TLRs is related to the circadian rhythm,and detected increased expression of TLR9 in Japanese medaka embryonic cells(OLHdr R-e3)with high expression of Bmal1 and Clock genes.Furthermore,in a TLR9-dependent mouse model of sepsis,disease severity was found to depend on the timing of sepsis induction,consistent with day-to-day changes in TLR9 expression and function.In isolated macrophages,LPS induces a phase shift of the circadian rhythm through TLR4 and suppresses the peak expression of the core circadian rhythm gene Bmal1.Melatonin(MT)is an indole substance secreted by the pineal gland,which can play an antioxidant and anti-inflammatory role.Studies have shown that MT can inhibit renal inflammation and play a renal protective role,but its efficacy and mechanism in DKD need to be further studied.Methods: Part I: Intervention of different concentrations of melatonin in high glucosestimulated rat glomerular mesangial cells for 48 hours.Western blot was used to detect the protein expressions of CLOCK,BMAL1,DEC1,TLR2,My D88 and NF-κB in the total proteins of each group.The protein expression of TGFβ1、 Collagen Ⅳand FN in the total protein of each group were detected by Western blot.The m RNA transcription and expression of TGFβ1,Collagen Ⅳand FN in the cells of each group were determined by Quantitative Real-time PCR;the expression of IL-1β and IL-6 in the culture supernatant of the cells in each group was detected by ELISA kit.Part Ⅱ: After one week of adaptive feeding in SPF-grade male mice,8-week-old T2 DM db/db mice were randomly divided into: db/m group,db/db group,and db/db+MT group.Mice in each group were kept in an environment with alternating light and dark for 12h-12 h.The light was turned on at 7AM in the morning,which was recorded as ZT0,and the light was turned off at 7PM in the evening,which was recorded as ZT12.In the db/db+MT group,melatonin was added to drinking water every day(ZT12-ZT0 the next day)(MT was dissolved in absolute ethanol,and then diluted with normal saline to make it into a normal saline solution containing 0.2% MT).The db/m group and the db/db group were given normal saline solution containing the same amount of ethanol,and the three groups were given ordinary feed.Dosing for 12 consecutive weeks.The mice were weighed once a week,their blood glucose was detected once a month,and their urine was collected,and the urine albumin and urine NGAL-related indicators were detected by enzyme-linked immunosorbent assay,and the urine albumin/urine creatinine ratio was determined.After 20-week-old mice were anesthetized,blood was collected by enucleating the eyeball,and kidney tissue was collected after cardiac perfusion.After the collected blood samples were processed,the indexes related to creatinine and blood urea nitrogen were detected by enzyme-linked immunosorbent assay.The prepared tissue sections were stained with HE,PAS and Masson.The expression levels of CLOCK,BMAL1,DEC1,TLR2 and NF-κB were detected based on immunohistochemistry.The protein expressions of CLOCK,BMAL1,DEC1,TLR2,My D88,NF-κB,TGFβ1,Collagen Ⅳ and FN in renal tissue were determined by Western blot.The expression of TGFβ1,Collagen Ⅳ and FN m RNA in renal tissue was detected by real-time quantitative PCR.ELISA kits were used to detect the expression of IL-1β and IL-6 in renal tissue homogenate of each group.Part Ⅲ: Chromatin immunoprecipitation(Ch IP)combined with PCR technique was used to identify the binding of transcription factors CLOCK and DEC1 to TLR2promoter-specific sequences under in vivo conditions.The effects of transcription factors CLOCK and DEC1 on the transcriptional activity of TLR2 promoter were detected by dual-luciferase reporter technology.The DEC1 si RNA was transfected with liposome,and the transfection efficiency was verified by real-time quantitative PCR.The protein expression levels of DEC1,TLR2,My D88,and NF-κB in each group of cells were detected by Western blot.Results: Part Ⅰ: The expression levels of circadian clock genes CLOCK and BMAL1 in glomerular mesangial cells in the HG group were significantly lower than those in the NG group(P<0.05),and the expression level of DEC1 was significantly increased compared with the NG group(P<0.05).Compared with the NG group,the expressions of TLR2,My D88,NF-κB,TGFβ1,Collagen Ⅳ and FN in the membrane cells were significantly increased,and the levels of IL-1β and IL-6 in the cell culture supernatant were increased.Melatonin effectively increased the expression levels of CLOCK and BMAL1 in mesangial cells stimulated by high glucose,decreased the expression level of DEC1,and reduced the production of TLR2,My D88,and NF-κB.Melatonin can downregulate the high expression of the cytokine TGF-β1 caused by high glucose,and can also inhibit the production of collagen Ⅳ and FN in the extracellular matrix,and reduce the levels of IL-1β and IL-6 in the cell culture supernatant.Part II: Melatonin reduces urinary ACR and urinary NGAL in db/db mice,improves renal pathological changes,reduces renal pathological structural changes such as mesangial matrix hyperplasia,and significantly reduces body weight and blood sugar in db/db mice.Melatonin can up-regulate the expression levels of clock genes CLOCK and BMAL1 in the kidney tissue of type 2 diabetic db/db mice,reduce the expression level of DEC1,down-regulate the TLR2/My D88/NF-κB pathway,reduce the inflammatory state in the kidney tissue and reduce the type Ⅳ The production of collagen and FN plays a protective role in the kidneys.Part III: Chromatin immunoprecipitation(CHIP)results showed that both CLOCK and DEC1 could bind to the TLR2 promoter.The results of dual luciferase assay showed that the transcription factor DEC1 could significantly up-regulate the luciferase expression level of p GL3-TLR2-wt,while the transcription factor CLOCK had no significant effect on the transcriptional activity of the wild-type TLR2 promoter and mutant TLR2 promoter.In the experiment of liposome transfection of DEC1 si RNA,the expression of DEC1 m RNA in the HG+DEC1-si RNA group was significantly downregulated compared with the HG group after 48 hours of intervention in the four groups(P<0.01).Compared with the HG group,the protein expressions of DEC1,TLR2,My D88 and NF-κB were significantly down-regulated in the HG+DEC1-si RNA group and the HG+MT group(P<0.05).There was no significant difference in the above indexes between the HG group and the HG+si NC group.Conclusion: Part Ⅰ: Rat glomerular mesangial cells have abnormal clock gene expression under high glucose state,TLR2/My D88/NF-κB signaling pathway is activated,the secretion of IL-1β,IL-6 and other inflammatory factors increases,and TGFβ1 signaling The expression of the pathway is up-regulated,resulting in increased secretion of Collagen Ⅳ and FN in the extracellular matrix of rat glomerular mesangial cells.Melatonin intervention can effectively regulate clock genes,inhibit the above signaling pathways to a certain extent,and reduce the glomerular glomerulus of rats.Secretion of extracellular matrix by mesangial cells.Part II: Melatonin can reduce urinary ACR and urinary NGAL in db/db mice,improve renal pathological changes,reduce mesangial matrix hyperplasia,and reduce body weight and blood glucose levels in db/db mice.Melatonin may regulate the renal tissue clock gene of type 2 diabetes db/db mice,down-regulate the TLR2/My D88/NF-κB signaling pathway,thereby inhibiting the activation of TGFβ1 signaling pathway,reducing the inflammatory state and fibrosis in renal tissue.Kidney protection.Part III:The transcription factor DEC1 may upregulate the TLR2/My D88/NF-κB signaling pathway by binding to the TLR2 promoter and activating its transcription,thereby activating the activation of the TGFβ1 signaling pathway,leading to renal inflammatory state and fibrosis,resulting in renal injury.