Krill Oil Turns Off TGF-β1 Profibrotic Signaling In Prevention Of Diabetic Nephropathy

BackgroundDiabetes mellitus(DM)is a nutritional disease that poses a severe threat to human health.Owing to the change in dietary pattern and lifestyle,the incidence of DM is increasing year by year,with type 2 diabetes mellitus(T2DM)accounting for 90%of the diabetic population.This has made DM a major problem of the nation’s public health,which urgently needs to be solved.DM results in several vascular complications which are the main cause for morbidity and mortality.Diabetic nephropathy(DN)is a serious microvascular complication of DM.As the main cause for end-stage renal disease,DN results in high morbidity and mortality,posing a severe threat to diabetic patients,health and leaving a heavy burden to social economy.To date,few effective approaches have been developed to prevent the pathogenesis and progression of DN.Therefore,it is important to explore efficient nutritional approaches for the intervention of DN,which is of scientific importance and social value.Krill oil(KO)is a functional food extracted from Antarctic krill,and is rich in functional components such as omega-3 polyunsaturated fatty acids,astaxanthin and phospholipids.KO was reported to have antiinflammatory and anti-oxidative effects.However,the effect of KO on the intervention of DN has not been studied.The present study aimed to evaluate the preventive effect and molecular action of KO on experimental DN using a mouse model of T2DM.Objectives1.Assessment of the preventive effect of KO on experimental DN.2.Exploration of the molecular mechanism of KO on experimental DN.Methods1.Assessment of the preventive effect of KO on experimental DNA mouse model of T2DM was established by intraperitoneal injection of streptozotocin(STZ)in combination with feeding with high-fat diet(HFD).Male C57BL/6N mice aged 7 weeks were culled for the experiment.After one-week adaptive feeding,the T2DM model mice were intraperitoneally injected with STZ(50 mg/kg),and the non-diabetic control mice(control,Ctrl group)with sodium citrate solution(0.1 mol/L),for 5 consecutive days.One week after the last injection,blood glucose levels were recorded,with a value≥13.89 mmol/L considered as diabetic.The T2DM model mice were then randomly divided into the diabetic group(DM)and the KO intervention group(DM+KO).The Ctrl,DM and DM+KO groups were immediately fed with normal chow diet,HFD and HFD containing 1.5%KO,respectively.For every 2 days,the food was renewed,and food intake and body weight were recorded.For every 4 weeks,blood glucose level was monitored,and urine was collected.Twenty-two weeks of post the onset of DM,glucose tolerance test was performed.Twenty-four weeks post DM onset,the mice were sacrificed,with the blood and kidneys harvested for analysis.1.1 The levels of urinary microalbumin and creatinine were determined by ELISA kits,with urinary microalbumin to creatinine ratio calculated.1.2 The renal pathological injuries of the mice were assessed by hematoxylin-eosin staining,periodic acid-Schiff staining and Masson’s trichrome staining.1.3 The mRNA levels of renal inflammatory,oxidative and fibrotic genes were measured by quantitative real-time PCR.1.4 The protein levels of renal inflammatory,oxidative and fibrotic genes were determined using Western blot.1.5 Immunohistochemical staining was used to locate specific proteins in the kidney.2.Investigation of KO’s action in the prevention of DNMouse mesangial cells(MMCs)were stimulated by high glucose(30 mmol/L)to establish a model of MMCs injury,accompanied by simultaneous treatment with either glycerol(the solvent of KO)or KO,for 48 hours.In detail,the experimental groups were:the normal glucose(5.5 mmol/L)group,the normal glucose+mannitol group,the high glucose group,the high glucose+glycerol group,and the high glucose+KO group.2.1 After RNA extraction,RNA-sequencing analysis was performed to screen major molecular pathways and targets modulated by KO.2.2 Quantitative real-time PCR and Western blot were used to verify the mRNA and protein levels of the screened-out key molecules.2.3 According to the screening and validation,MMCs were stimulated with transforming growth factor-beta 1(TGF-β1,10 ng/mL),with simultaneous treatment with glycerol or KO,for 48 hours.2.4 The expression of the key factors within TGF-β1 signaling pathway,such as TGF-β1 receptor 1,TGF-β1 receptor 2,SMAD4,SMAD7,SMAD2,SMAD3,p-SMAD2 and p-SMAD3 were measured by quantitative real-time PCR and Western blot.Moreover,the nuclear translocation of SMAD2/3 was detected by immunofluorescent staining.2.5 The effect of KO on the expression of the key factors of TGF-β1 signaling pathway was further verified in the kidneys,using quantitative real-time PCR,Western blot and immunohistochemical staining.Results1.Although KO did not lower blood glucose levels and improve glucose intolerance,it significantly decreased urinary microalbumin to creatinine ration,and prevented mesangial matrix accumulation,glomerular hypertrophy and fibrosis,and inhibited renal expression of inflammatory,oxidative and fibrotic genes in the diabetic mice.2.KO significantly regulated TGF-β1 signaling pathway in the high glucose-stimulated MMCs,as indicated by RNA-sequencing.Further experiments verified that KO could inhibit the TGF-β1-induced expression of fibrotic genes.Mechanistically,KO prevented the TGF-β1triggered phosphorylation and nuclear translocation of SMAD2/3,and activated the expression of the Smad7 gene,the protein product of which inhibits SMAD2/3 phosphorylation and activation.3.KO inhibited the phosphorylation of SMAD2/3,increased the expression of Smad7 in the kidneys of the diabetic mice.Conclusions1.KO could effectively prevent the DM-induced renal dysfunction,pathological injuries,and expression of inflammatory,oxidative and fibrotic genes.2.KO may inhibit the DM-induced renal fibrosis via the activation of Smad7 gene expression,and the inhibition of SMAD2/3 phosphorylation and nuclear translocation.