The Role Of Sweet Taste Receptors In The Activation Of ROS-NLRP3 Inflammasome Signaling In Diabetic Kidney Disease

Objective:Diabetic nephropathy(DN)is a microvascular complication associated with severe diabetes mellitus(DM).DN is the most common cause of end-stage renal disease,patients with diabetes have approximately one-third rate to develop to DN.There are many factors involved in the pathogenesis of DN.Many studies have shown that long-term high glucose stimulating and other metabolic disorders can induce excessive production of reactive oxygen species(ROS)in mitochondria,and ROS is an important promoter of DN,Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3)inflammasome signaling pathway is activated to induce the mature and secreted of Cysteine-aspartic acid protease1(Caspase1)and interleukin-1β(IL-1β)and involved in the Different pathological processes of diabetic kidney disease and play an important role.However,up to now,the mechanism by which high glucose induces the activation of the ROS-NLRP3 inflammasome signaling pathway has not yet been fully elucidated as to whether the natural ligand glucose of the sweet receptor can affect ROS-NLRP3 inflammasome signaling pathway activation through sweet taste receptors(STRs)remains to be further studied.Studies have shown that STRs are mainly composed of taste receptor type 1,member 2(T1R2)and member3(T1R3)and widely expressed in the oral tongue and various organs other than the oral cavity as a chemical Sensors involved in sugar absorption,uptake,distribution,storage and innate immunity,and obesity,diabetes and cardiovascular disease and other diseases are closely related to the occurrence and development.Therefore,in our study,the expression changes of T1R2,T1R3,ROS,NLRP3,Caspase1 and IL-1β in mouse mesangial cells and human proximal convoluted tubule epithelial cells at different times and different concentrations under high-glucose stimulating,and the expression of IL-1β in the cell culture supernatant was observed.The changes of key factors in the inflammasome of ROS-NLRP3 were observed again after the cells were transfected with T1R2/T1R3-si RNA to interfere the expression of STRs,aiming to investigate the effect of high glucose on the expression of T1R2 and T1R3 in mouse mesangial cells and human proximal tubule epithelial cells and the regulation of STRs on ROS-NLRP3 inflammasome signaling pathway,providing a new idea for the prevention and treatment of DN.Methods: 1.Effect of diabetic kidney disease and sweet receptor inhibitor on the renal sweet receptor and ROS-NLRP3 inflammation in mice;(1)Twenty male C57BL/6 mice(body weight(20 ± 3)g)were randomLy divided into two groups(n = 10):(1)NC group: continue to feed with normal maintenance feed;(2)Type 2 diabetes mellitus(T2DM group):rats were fed with high fat and sucrose diet continuously,and at 8 weeks,injected with 100 mg/kg Streptozocin(STZ)intraperitoneally;Twenty mice injected with STZ were monitored for blood glucose,the modeling of T2 DM was successfully made when glucose≥16.7 mmol/L.Urinary albumin-creatinine ratios(ACRs)were collected every four weeks after successfully modeling.Mice were sacrificed in the 12 weeks after successfully modeling,blood was taken from the heart for detection of blood urea nitrogen(BUN)and serum creatinine(Scr)using automatic biochemical analyzer.Take the left kidney,formaldehyde fixed and paraffin embedded,and then immunohistochemistry was used to detect the expression of T1R2 and T1R3.Take the right kidney,extract mRNA and protein after tissue homogenate,detect the protein expression of T1R2,T1R3,NLRP3,Caspase1,IL-1β with Western-blot,use qRT-PCR to detect the expression of T1R2,T1R3 mRNA.(2)The effect of lactisole on the inflammatory of ROS-NLRP3: Thirty male mice(ibid.)Were randomLy divided into three groups(n = 10):(1)NC group: fed with normal maintenance feed and normal drinking water continuously;(2)Type 2 diabetes mellitus(T2DM group): The rats were fed with high-fat,high-sugar and normal drinking water,and at 8 weeks;intraperitoneal injection of 100mg/kg streptozotocin(Streptozocin,STZ).(3)Type 2diabetes millitus,T2 DM + lac group: The rats were fed with high-fat and high-sucrose diet for 8 weeks,and were injected intraperitoneally with100mg/kg STZ and fed 0.04mg/mL lac drinking water simultaneously.Thirty mice after STZ injection were monitored for blood glucose,the T2 DM model was successfully established when glucose≥16.7mmol/L.The lac binding and competition inhibition test were performed in 12 weeks after the successfully modeling.The effects of diabetic kidney disease and lac on the expression of the sweet receptor of kidney in mice were observed with mouse PET/CT.2.Effect of high glucose and sweet receptor inhibitors on the expression of sweet taste receptor in mesangial cells and the inflammatory response of ROS-NLRP3 in mice:(1)Cultured mouse glomerular mesangial cells(SV40)in vitro,with high glucose as a stimulating factor,is divided into three groups:(1)Normal control group(NC group): using 5.6 mmol/L glucose medium;(2)Osmotic pressure group(OP group): using 5.6 mmol/L glucose medium + 24.4 mmol/L mannitol;(3)High glucose group(HG group): Culture medium divided into 10 mmol/L glucose(HG1 group),20 mmol/L glucose(HG2 group)and 30 mmol/L glucose(HG3 group).The cells were cultured for 6h,12 h,after that,use Western-blot to detect the protein expression of T1R2,T1R3,NLRP3,Caspase1,IL-1β;use qRT-PCR to detect the expression of T1R2,T1R3 mRNA;use ELISA to detect the concentration of culture supernatant IL-1β.(2)Study of lactisole(lac): The best high glucose concentration(30mmol/L)and time(24h)were screened out and then subdivided into groups:(1)NC group: medium containing 5.6 mmol/L glucose;(2)HG3 group: medium containing 30 mmol/L glucose;(3)HG3+ lac group: using 30 mmol/L glucose medium + 3 mmol/L lac(L1 group)or 5 mmol/L lac After culturing the cells for 24 hours,the above parameters were detected again by Western-blot,qRT-PCR and ELISA.4.Statistical Analysis: Using SPSS 24.0 statistical software to analyze the data processing.Metric data in line with the normal distribution of mean± standard deviation((?)±s)said the use of single-factor analysis of variance between groups of mean,multiple comparison between groups using LSD-t method.We define P <0.05 as a statistically significant difference.Results:(1)Compared with NC group,prolonged high glucose stimulation(> 12h)could down-regulate the expressions of protein and mRNA of sweet receptor(T1R2,T1R3)in SV40 and HK-2 cells and increase the production of ROS in SV40 and HK-2 cells;NLRP3,Compared with NC group,the expression of Caspase1,IL-1β protein and mRNA in T2 DM group was significantly lower than that in NC group(P<0.05)T1R2,T1R3)protein and mRNA expression was down-regulated,while NLRP3,Caspase1,IL-1β protein and mRNA expression was up-regulated.(2)Compared with HG3 group,HG3 + lac group could partly reverse the down-regulation of the protein and mRNA expression of the sweet taste receptor(T1R2,T1R3)in SV40 and HK-2,and reduce the ROS production in SV40 and HK-2 cells,The expression of NLRP3,Caspase1,IL-1βprotein and mRNA in T2 DM + lac group was significantly higher than that in T2 DM + lac group(P <0.05),while NLRP3,Caspase1,IL-Downregulation of mRNA expression..(3)Compared with NC group,the expression of protein and mRNA of sweet receptor in HK-2 cells was induced by high glucose(0-12h)(P <0.05),and was significantly increased at 12mmol/L Enhanced;T1R2/T1R3 si RNA transfected cells can successfully inhibit T1R2/T1R3 expression.Compared with NC + sicontrol group,the intracellular ROS production,Caspase1 and IL-1β protein expression in NC + si T1R2,si T1R3 and si T1R3/T1R3 groups were significantly increased(P <0.05);compared with HG3 + sicontrol group,HG3 + si T1R2 The intracellular ROS production,Caspase1 and IL-1β protein expression in si T1R3 and si T1R3/T1R3 groups also increased significantly(P <0.05).Conclusion:(1)The expression of STRs(T1R2 T1R3)in vivo and in vitro was significantly decreased under the regulation of hyperglycemia in a time and dose dependent manner.(2)STRs inhibitor lactisole could significantly reduce intracellular ROS production and reverse the activation of signaling pathways of ROS-NLRP3 inflammasome stimulated by high glucose in GMCS in a dose-dependent manner(P<0.05).These results support the hypothesis that hyperglycemia can induce ROS-NLRP3 inflammasome signaling pathway activation,in part,through the sweet receptor,suggesting that STRs may serve as a new target for DN therapy.