MCC950 And Fermented Cordyceps Sinensis Ameliorated Diabetic Kidney Injury By Downregulating NLRP3/Caspase-1/IL-1β Pathway

Introduction: Diabetic nephropathy(DN)is the most common microvascular complication in diabetic patients and the leading cause of end-stage renal disease(ESRD)worldwide.The pathogenesis of DN is complex,genetic and environmental factors are involved.The classic view of metabolic and hemodynamic alterations as the main causes of kidney injury in DN has been transformed,while more viewpoints hold that chronic low-grade inflammation is the crucial factor leading to the progression of DN.Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing protein 3(NLRP3)is a pattern recognition receptor that is activated by hyperglycemia.NLRP3 combined with ASC,recruits pro-caspase-1 to form the inflammasome and turned into caspase-1 by hydrolysis.Active caspase-1 induces pro-IL-1β maturation and IL-1β secretion;and cleave GSDMD to trigger a form of programmed cell death called pyroptosis.Unresolved inflammatory reactions make kidney damage progressing continuously and then develop into renal fibrosis,which eventually leads to ESRD.MCC950 is a potent and selective small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations.MCC950 was reported with early promise for treatment of several inflammatory diseases such as atherosclerosis,type 2 diabetes,rheumatoid arthritis and asthma;however,its efficacy in DN requires further investigation.Cordyceps sinensis(Dong Chong Xia Cao),a traditional Chinese medicine known as Chinese caterpillar fungus,has potential immunomodulatory,antioxidant,anti-inflammatory,antitumor and antiviral properties.Fermented Cordyceps sinensis as a substitute for costly natural Cordyceps sinensis,has been widely in clinical use.Fermented Cordyceps sinensis(Cs-4)have been recorded in Chinese Pharmacopoeia and have relatively comprehensive quality standards.The active chemical components of Cs-4 is complex;however,its efficacy and mechanisms in DN requires further researches.Methods: The first part:According to the enrollment and exclusion criteria,6 patients with type III diabetic nephropathy by renal biopsy and 6 patients with the control group were enrolled.The expression of NLRP3,caspase-1 and IL-1β was detected by immunohistochemistry.High-glucose induced rat glomerular mesangial cells were treated with various concentrations of MCC950 for 48 hours.Western blot was used to measure the expression of NLRP3 、 caspase-1 and IL-1β in mesangial cells,and immunofluorescence was used to detect caspase-1.Western blot was used to measure the expression of TGF-β1,collagen I and α-SMA in mesangial cells.Real-time q PCR was used to measure the expression of TGF-β1,collagen I and FN m RNA.The second part:After one week of adaptive feeding,SPF male eight-week-old type 2 db/db mice were randomly divided into MCC950 treated group and non-treated group,with db/m mice as normal control group.For the MCC950 treated group(db/db+MCC950),db/db mice were treated with 10 mg/kg of MCC950 twice per week intraperitoneally.The control(db/m)and untreated groups(db/db)received an equal volume of vehicle(saline)for 12 weeks.Body weight(BW)was measured weekly,and blood glucose levels were measured every 4 weeks.Mice were housed in individual metabolic cages,and urine samples were collected every 4 weeks.Urinary albumin and neutrophil gelatinase-associated lipocalin(NGAL)were determined by ELISA.Urinary creatinine was detected by creatine oxidase method.Urinary albumin concentration versus creatinine concentration ratio(ACR,μg/mg)was calculated.When the twenty-week-old mice were sacrificed,all mice were anesthetized,and blood samples were collected by posterior orbital venous plexus bleeding.Renal cortical samples were harvested for subsequent studies after cardiac perfusion.Serum samples were used to measure serum creatinine and blood urea nitrogen(BUN)on an automatic biochemical analyzer.Paraffin sections were stained with hematoxylin-eosin(HE),periodic acid-Schiff(PAS)and Masson trichrome.The ultrastructure of kidney was examined using transmission electron microscope(TEM).The expression of NLRP3、caspase-1、IL-1β、FN、α-SMA and podocin were detected by immunohistochemistry.The protein expression of NLRP3、caspase-1、IL-1β、GSDMD、TGF-β1、collagen I、α-SMA and podocin in renal cortices were determined by Western blot.The m RNA expression of TGF-β1、collagen I and FN in renal cortices were detected by real-time PCR.The third part: After one week of adaptive feeding,SPF male eight-week-old type 2 db/db mice were randomly divided into Cs-4 treated group and non-treated group,with db/m mice as normal control group.For the Cs-4 treated group(db/db+Cs-4),db/db mice received daily gavage of fermented Cordyceps sinensis(Cs-4,1g/kg).The control(db/m)and untreated groups(db/db)received an equal amount of distilled water as the vehicle.The treatment lasted from the age of 8 weeks to 20 weeks.Body weight was measured weekly,and blood glucose levels were measured every 4 weeks.Mice were housed in individual metabolic cages,and urine samples were collected every 4 weeks.Urinary albumin and NGAL were determined by ELISA.Urinary creatinine was detected by creatine oxidase method.Urinary ACR was calculated.24-hour urine volume of the twenty-week-old mice were recorded.When the twenty-week-old mice were sacrificed,all mice were anesthetized,and blood samples were collected by posterior orbital venous plexus bleeding.Renal cortical samples were harvested for subsequent studies after cardiac perfusion.Serum samples were used to measure serum creatinine and BUN by an automatic biochemical analyzer.Paraffin sections were stained with HE,PAS and Masson.The ultrastructure of kidney was examined using TEM.The expression of NLRP3 、 caspase-1 、 IL-1β 、 FN 、 α-SMA and podocin were detected by immunohistochemistry.The protein expression of NLRP3、caspase-1、IL-1β、GSDMD,TGF-β1、collagen I、α-SMA and podocin in renal cortices were determined by Western blot.The m RNA expression of TGF-β1、collagen I and fibronectin in renal cortices were detected by real-time PCR.Results: The first part:Compared to the control group,NLRP3/Caspase-1/IL-1β pathway was up-regulated in kidney tissue of patients with diabetic nephropathy and high-glucose induced mesangial cells.MCC950 effectively inhibited the activation of NLRP3,and reduced the production of active caspase-1 and active IL-1β in high-glucose induced mesangial cells.MCC950 significantly reduced the expression of cytokine TGF-β1,the production of extracellular matrix protein collagen I and FN,and α-SMA in high-glucose induced mesangial cells.The second part:MCC950 had no effect on body weight and blood glucose levels in type 2 diabetic db/db mice.MCC950 reduced serum creatinine,urinary ACR and urinary NGAL in db/db mice,attenuated pathological changes of glomerular hypertrophy,mesangial expansion with increased matrix and tubular dilatation in db/db mice,alleviated renal ultrastructural changes such as thickened glomerular basement membrane(GBM)、foot process fusion and disappearance of endothelial cell window pore structure.MCC950 inhibited the activation of NLRP3,and reduced the production of active caspase-1 and active IL-1β and the expression of GSDMD as a marker of pyroptosis.MCC950 reduced the expression of TGF-β1、FN、collagen I and α-SMA,and increased the expression of podocin as a marker of podocyte.The third part: Cs-4 had no effect on body weight in type 2 diabetic db/db mice.Cs-4 decreased blood glucose levels of db/db mice only at 16 weeks of age.Cs-4 reduced serum creatinine,24-hour urine volume,urinary ACR and urinary NGAL in db/db mice,attenuated pathological changes of glomerular hypertrophy,mesangial expansion with increased matrix and tubular dilatation in db/db mice,alleviated renal ultrastructural changes such as thickened GBM、foot process fusion and disappearance of endothelial cell window pore structure.Cs-4 down-regulated NLRP3 expression,and reduced the production of active caspase-1 and active IL-1β and the expression of GSDMD.Cs-4 reduced the expression of TGF-β1、FN、collagen I and α-SMA,and increased the expression of podocin.Conclusion: The first part:NLRP3/Caspase-1/IL-1β pathway was up-regulated in kidney tissue of patients with diabetic nephropathy and high-glucose induced mesangial cells.MCC950 effectively inhibited NLRP3,and decreased the production of cytokines、extracellular matrix accumulation and the transformation of mesangial cells into myofibroblasts by down-regulating NLRP3/Caspase-1/IL-1β pathway in high-glucose induced mesangial cells.The second part: MCC950 reduced urinary ACR and urinary NGAL,improved renal function and pathological changes,reduced mesangial expansion with increased matrix,and alleviated renal ultrastructural changes such as thickened GBM、foot process fusion and disappearance of endothelial cell window structure in db/db mice.MCC950 ameliorated podocyte injury without affecting body weight and blood glucose levels.The effect of MCC950 was independent of body weight and blood glucose levels.MCC950 inhibited NLRP3 in kidney tissue of type 2 diabetic db/db mice with down-regulating NLRP3/Caspase-1/IL-1β pathway,and decreased active GSDMD transformation to alleviate renal inflammation and pyroptosis,which played the role of kidney protection.The third part: Cs-4 reduced 24-hour urine volume,urinary ACR and urinary NGAL;improved renal function and pathological changes;reduced mesangial expansion with increased matrix;alleviated renal ultrastructural changes such as thickened GBM,foot process fusion and disappearance of endothelial cell window structure in db/db mice.Cs-4 ameliorated podocyte injury.Cs-4 maybe down-regulate NLRP3/Caspase-1/IL-1β pathway in kidney tissue of type 2 diabetic db/db mice,to alleviate renal inflammation and pyroptosis.The anti-inflammatory effect of Cs-4 in DN played the role of kidney protection.