| Background and purpose:Diabetic nephropathy（DN）is the most common microvascular complication of diabetes.Although DN is characterized by hemodynamic and metabolic changes caused by hyperglycemia,chronic inflammation mediated by macrophages is widely regarded as an important event in the pathological process of DN.Therefore,any way to inhibit macrophage immune inflammation will bring hope to the treatment of DN.Exploring specific inflammatory targets and screening preventive and therapeutic drugs have important clinical significance.Macrophages have the characteristics of strong phenotypic plasticity.M1 macrophages mainly secrete cytokines such as Interleukin-1β（IL-1β）and tumor necrosis factorα（TNF-α）,which promote the occurrence and development of inflammation.A class of non-coding transcripts longer than 200bp,collectively known as long non-coding RNA（Lnc RNA）,have been shown to regulate inflammatory factors and macrophage phenotypes by controlling downstream signals,participate in promoting inflammation and fibrosis,and are closely related to chronic inflammation associated with diabetes.At the same time,it was found that Kruppel-likefactor4（KLF4）was involved in the regulation of persistent inflammatory injury,which was specifically reflected in promoting the functional transformation of macrophages,inhibiting the phosphorylation of NFκB in the inflammatory pathway and reducing the release of pro-inflammatory factors.KLF4 is an evolutionarily conserved transcription factor with zinc finger structure,and it also shows the effect of anti-inflammation and anti-fibrosis.the expression of KLF4 in diabetic renal tissue is significantly lower than that in normal renal tissue.Therefore,to explore drugs that can change the direction of macrophage inflammation is an urgent need to alleviate the disease of DN.Wogonoside（WG）belongs to bioactive flavonoids and is the metabolite of WG.It has the effects of antioxidation,anti-inflammation,anti-virus and anti-cancer.At present,it has not been reported whether DNM3OS and KLF4 are involved in diabetic renal inflammation,and whether WG can inhibit the progression of DN inflammation and its mechanism.In this study,renal tissue and serum samples of patients with clinical DN were collected to observe the expression of target gene and inflammatory factors,macrophages were used to simulate the internal environment of diabetic nephropathy in vitro,and si RNA technique was used to knock down DNM3OS gene,to explore the specific mechanism of DNM3OS-induced signal pathway in high glucose environment,and to observe the effect of WG on related signal pathway proteins and inflammatory factors in macrophages.In vitro,db/db mice were used to observe the expression of target gene and the effect of WG on renal inflammation in DN.Method:1.From June 2019 to June 2020,20 cases of normal renal tissue adjacent to cancer in the Renal Pathology Center of the first affiliated Hospital of Anhui Medical University were collected as control group,and 35 cases of renal tissue from patients with DN were selected as DN group.The renal tissues of the two groups were embedded,sliced and stained,analyzed by microscope and observed by electron microscope,and the related indexes of pathological damage in the two groups were calculated.The general indexes of glycosylated hemoglobin,serum creatinine,blood pressure,urea nitrogen,uric acid,glomerular filtration rate and 24-hour urinary albumin were counted.The levels of Interleukin1β（IL-1β）and Tumor necrosis factor-α（TNF-α）in serum samples of patients with DN could be detected by Enzyme linked immunosorbent assay,and the serum samples of healthy volunteers were taken as normal controls.Fluorescence in situ hybridization and RT-PCR were used to detect the expression of DNM3OS in renal tissues of the two groups.Immunohistochemistry was used to detect the expression of CD68 and TNF-αin renal tissue of the two groups.2.Mouse RAW264.7 macrophages were selected for the experiment.Firstly,the intervention concentration range of WG was screened by CCK8,and the experiment was divided into LG group(5.5mmol?L-1glucose medium),D group(30 mmol?L-1mannitol medium),HG group(30mmol?L-1glucose medium),LG+WG50 group(5.5 mmol?L-1glucose+μmol?L-1WG),HG+WG12.5 group(30mmol?L-1glucose+12.5μmol?L-1WG).HG+WG25 group(30mmol?L-1glucose+25μmol?L-1WG)and HG+WG50 group(30 mmol?L-1glucose+50μmol?L-1WG).The content of inducible nitric oxide synthase（i NOS）in LG group,LG+W50group,HG group and HG+WG50 group was detected by immunofluorescence（IF）.The protein expression of phosphorylated nuclear factor（NFκB-NFκB-pp65）,KLF4,i NOS,TNF-αand IL-1βwas detected by Western blot.The m RNA expression of KLF4,i NOS,TNF-αand IL-1βwas detected by RT-q PCR.The content of IL-1βand TNF-αin the supernatant of each group was detected by ELISA.The expression of DNM3OS in each group was detected by RT-q PCR,then silenced DNM3OS gene was transfected by si RNA,and the highest transfection efficiency reagent was screened by Western blot.The experimental group was divided into LG group(5.5mmol?L-1glucose medium),LG+si RNA control group(5.5mmol?L-1glucose medium+si RNA),LG+WG50 group (5.5mmol?L-1glucose+50μmol?L-1WG),HG group(30mmol?L-1glucose medium).HG+si DNM3OS group(30 mmol?L-1glucose+si DNM3OS)and HG+WG50 group(30 mmol?L-1glucose+50μmol?L-1glucose).The protein expression of NFκB-pp65,KLF4,TNF-αand IL-1βwas detected by Western blot,and the m RNA expression of KLF4,TNF-αand IL-1βwas detected by q PCR.3.After feeding several male db/m and db/db mice for 6 weeks,they were divided into four groups:db/m group,db/m+WG50 group,db/db group,db/db+WG12.5 group,db/db+WG25 group and db/db+WG50 group.Drug intervention was carried out for 12 weeks.At the end of feeding,the24-hour urine of mice in each group was collected by metabolic cages,and then the eyeball was removed to take the serum,and the kidney tissue was weighed and the kidney weight ratio was recorded.The degree of renal pathological injury was observed by PAS staining light microscope and the ultrastructure was observed by transmission electron microscope.IHC was used to detect the expression of inflammatory markers CD68 and TNF-αin db/m group,db/m+WG50 group and db/db group.Western blot was used to detect the protein expression of NFκB-pp65,KLF4,i NOS,TNF-αand IL-1βin renal tissue of mice in each group.RT-PCR was used to detect the m RNA expression of KLF4,i NOS,TNF-αand IL-1βin renal tissue of mice in each group.RT-q PCR was used to detect the expression of DNM3OS in renal tissue of mice in db/m group and db/db group,and the content of IL-1βand TNF-αin serum of mice in each ELISA group.Reasult:1.The results of PAS showed that the glomerular Mesangial dilation index（p<0.01）,the degree of tubule injury and the diameter of glomeruli increased in DN group（p<0.01）.Electron microscope showed that the thickness of basement membrane increased in DN group（p<0.01）.The results of ELISA showed that the levels of IL-1βand TNF-αin serum of DN group were higher than those of Normal group（p<0 01）.IHC results showed that the expression of macrophage marker CD68 and inflammatory factor TNF-αin renal tissue increased in DN group（p<0 01）.The results of FISH showed that the expression of DNM3OS was increased in DN group（p<0.01）.2.Optimization of experimental conditions:CCK8 showed no effect on cell activity at 0-50μmol?L-1.Fluorescence microscope showed that the expression of i NOS increased in HG group and decreased in WG group（p<0.01）.The results of Western blot and RT-PCR showed that i NOS,TNF-α,IL-1βincreased and KLF4 decreased in HG group（p<0.01）,i NOS,TNF-αand IL-1βdecreased in a dose-dependent manner and KLF4increased in a dose-dependent manner after WG intervention（p<0.01）.After DNM3OS silencing,the expression of IL-1β,TNF-αand NFκB-pp65decreased and the expression of KLF4 increased in HG+si DNM3OS group（p<0.01）.In HG+WG50 group,the expression of IL-1β,TNF-αand NFκB-pp65 decreased and the expression of KLF4 increased（p<0.01）.The results of ELISA showed that IL-1βand TNF-αin HG group increased（p<0.01）,but decreased after drug intervention（p<0.01）.3.After 12 weeks,blood glucose,24-hour urinary protein metabolic rate and kidney weight in db/db group were significantly higher than those in db/m group（p<0.01）,while the related indexes in db/db plus drug group were lower than those in db/db group（p<0.01）.The results of PAS staining showed that the glomerular Mesangial dilation inde（p<0.01）x,the degree of renal tubule injury and the diameter of glomeruli increased in db/db group（p<0.01）.The pathological injury in db/db group was significantly improved（p<0.01）.The results of electron microscope showed that the thickness of basement membrane increased in db/db group and significantly improved in db/db plus drug group（p<0.01）.The results of ELISA showed that the levels of serum IL-1βand TNF-αin db/db group were higher than those in db/db group（p<0.01）,while those in db/db plus medicine group were lower than those in db/db group（p<0.01）.The results of Western blot and RT-PCR showed that NFκB-pp65,i NOS,TNF-αand IL-1βin renal tissue increased and KLF4 decreased in db/db group（p<0.01）,and NFκB-pp65,i NOS,TNF-αand IL-1βdecreased in a dose-dependent manner and KLF4 increased in a dose-dependent manner after WG intervention（p<0.01）.Conclusion:1.the expression of DNM3OS,Mesangial dilation index and serum TNF-αand IL-1βwere increased in patients with diabetic nephropathy.It was found that DNM3OS was closely related to the development of DN.2.High glucose could induce the activation of RAW264.7 cells and up-regulate the expression of DNM3OS,trigger the mechanism of intracellular inflammation,increase the release of TNF-αand IL-1βin cells and cell supernatants,increase the activation of p65 phosphorylation pathway,and inhibit the expression of transcription factor KLF4.WG can inhibit the activation of macrophages induced by high glucose,inhibit the expression of DNM3OS and the release of macrophage inflammatory cytokines TNF-αand IL-1β,and promote the expression of KLF4.Silencing DNM3OS gene can inhibit the activation of macrophages induced by high glucose,weaken the activation of p65 phosphorylation pathway,inhibit the release of macrophage inflammatory factors TNF-αand IL-1β,and promote the expression of KLF4.3.Mesangial dilation index,DNM3OS expression,i NOS、TNF-α、IL-1βexpression increased and KLF4 expression decreased in renal tissue of db/db mice.WG could inhibit the expression of DNM3OS、i NOS、TNF-α、IL-1β and increase the expression of KLF4 in renal tissue of db/db mice. |
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