| Diabetic kidney disease(DKD)is one of the most common and serious microvascular complications of diabetes mellitus,which seriously threatens human health.The activation of immune system and micro-inflammatory status have been the recognized pathogenesis of diabetic kidney disease.How to overcome the immune inflammation reaction and prevent the progress of DKD is an important topic in the field of prevention and treatment of DKD.Macrophages are the key immune inflammatory effector cells.Under the background of diabetes mellitus,macrophages infiltrated in the kidney and were mainly activated into the pro-inflammatory M1 type,which producing a variety of inflammatory cytokines,chemokines,pro-fibrotic factors,etc.,changing the local microenvironment of the kidney,causing glomerular damage,structural remodeling,hardening,tubule atrophy,interstitial inflammation and fibrosis.Infiltrating macrophages have undergone reprogramming of glucose metabolism(transition from resting to active metabolic state)from the resting state to the proinflammatory Ml-type classic activation state.M1 macrophages mainly rely on the fast-energy aerobic glycolysis pathway to obtain energy and intermediate media to exert pro-inflammatory effects.M2-type pyruvate kinase(PKM2)is a key enzyme for cellular metabolic reprogramming and the most important rate-limiting enzyme in glycolysis.Under physiological conditions,PKM2 exists in the cytoplasm in a tetrameric conformation with high kinase activity,and can form a complex with other sugar metabolizing enzymes,such as hexokinase,pyruvate dehydrogenase kinase,etc.,to drive the tricarboxylic acid cycle and regulate the cellular energy metabolism.Under micro-environmental stimulation,PKM2 tetramer dissociates into a low-activity dimer translocating to the nucleus,driving glycolysis and participating in macrophage activation.Nuclear PKM2 is a key determinant of M1 macrophage activation.Therefore,in the state of diabetes mellitus,it is of great theoretical and clinical significance to deeply study the mechanism of macrophage-related kidney damage based on immune metabolic enzyme points in order to screen out new targets for prevention and treatment of DKD.Traditional Chinese medicine(TCM)has irreplaceable advantages in delaying the progress of diabetic kidney disease.At present,the flowers of Abelmoschus Manihot(L.)Medic and preparations have become important drugs in the treatment for chronic kidney diseases,such as diabetic nephropathy and chronic nephritis.In this study,we established the db/db mice and RAW264.7 macrophages as the research object to explore the protective effect of total flavones of Abelmoschus Manihot(L.)Medic Flowers(TFA)on DKD kidney.To further study whether the mechanism was related to the infiltration and activation of macrophages in renal,and explore the possible targets.It provided a new experimental basis for the pharmacological mechanism of the flowers of Abelmoschus Manihot(L.)Medic in the treatment of DKD.Purpose:To observe the effect of TFA against renal inflammation and fibrosis by using molecular biology experimental techniques in db/db mice of DKD,and to explore whether the effect was related to the regulation of M2 pyruvate kinase(PKM2)or the infiltration and activation of M1 macrophages,which will provide scientific basis for the treatment of DKD by the flowers of Abelmoschus Manihot(L.)Medic.Methods:1.Animal experiment:according to the random number table method,twenty male db/db mice were divided into DKD model,low-dose(97.5mg/kg/d),medium-dose(195mg/kg/d)and high-dose(390mg/kg/d)mice groups,and db/m mice as the control group.The db/db mice in the treatment group were respectively treated with the corresponding dose of TFA for 16 weeks.The control group and the model group mice were treated with an equal volume of 0.5%sodium carboxymethylcellulose solution for 16 weeks.During the experiment,urine ACR was tested regularly.At the 24th week,specimens were collected.HE staining and Masson staining were used to observe renal morphological changes and the degree of renal fibrosis.Imrnunohistochemical staining was used to detect macrophage marker F4/80.The expressions of F4/80,PKM2,collagen ? and vimentin protein were detected by Western Blot.The mRNA expressions of TNF-?,IL-1?,iNOS and PKM2 were detected by RT-qPCR.The levels of serum TNF-? and MCP-1 were detected by ELISA2.Cell experiment:The CCK-8 method was used to detect the effect of different concentration gradients of TFA on the survival rate of RAW264.7 macrophages,which initially determined the appropriate concentration of scutellarin for subsequent pharmacodynamic experiments.After different sugar concentration gradients stimulated RAW264.7 macrophages,flow cytometry was used to screen the optimal sugar concentration for M1 macrophage activation.The RAW264.7 macrophages were divided into low-glucose group,high-glucose group,high-glucose+TFA group.Flow cytometry was used to analyze the effect of TFA on Ml macrophage activation from the RAW264.7 macrophages.Western blot was used to detect PKM2 protein expression and to explore dose-effect relationship.Result:1.Compared with the control group db/m mice:the levels of body weight and kidney index in the DKD model group were increased(P<0.01).The levels of urinary ACR in the DKD model group were increased(P<0.01).The levels of serum creatinine and urea nitrogen were increased(123.50±17.91 vs 13.40±1.36,15.22±0.63 vs 6.53±1.38,P<0.01).The renal pathological damage was obvious,and collagen fibers were deposited.Immunohistochemistry showed that the infiltration of F4/80 labeled macrophages increased in renal tissue(P<0.05).The protein expression of F4/80 and the mRNA levels of iNOS,TNF-? and IL-I? were increased(P<0.01).The protein expressions of Collagen III and Vimentin,the markers of renal fibrosis,were up-regulated(P<0.01).The levels of serum TNF-? and MCP-I were increased(P<0.01).The protein expression and the mRNA level of PKM2,a key enzyme of glucose metabolism reprogramming,were up-regulated(P<0.01).2.Compared with the DKD model group db/db mice:after the treatment of TFA,the kidney index of db/db mice had a certain improvement,but the difference was not statistically significant(P>0.05,the same as below).The level of urinary ACR decreased with the treatment of low,medium and high doses of TFA(P<0.05).The levels of serum creatinine and blood urea nitrogen were generally inhibited,and the differences of serum creatinine in the middle-dose group and blood urea nitrogen in the high-dose group were statistically significant(P<0.05)The difference of serum creatinine in the high-dose group was highly statistically significant(P<0.01).The protein expressions of Collagen III and Vimentin in kidneys of treatment mice group were inhibited to varying degrees.The expressions of Collagen III and Vimentin protein in the medium-dose and high-dose groups were highly significantly decreased(P<0.01).Immunohistochemistry of kidney tissues in treatment mice groups indicated that the infiltration of labeled F4/80 macrophages decreased in varying degrees,and the differences of the middle-dose and high-dose groups were statistically significant(P<0.05).The expression of F4/80 protein in the kidney of treatment mice group was decreased to different extents,and the difference in the medium-dose group was statistically significant(P<0.05),The difference in the high-dose group was highly significantly significant(P<0.01).The levels of TNF-?,IL-1?and iNOS mRNA in the kidney of the treatment mice group were significantly decreased(P<0.01).The levels of TNF-? and MCP-1 in the serum of mice in the treatment group were down-regulated(P<0.05),and the down-regulation in the high-dose group was more significant(P<0.01).The protein expression and mRNA level of PKM2 in the kidney of treatment mice group were decreased to varying degrees(P<0.05,P<0.01).The levels of body weight and kidney index of db/db mice in the treatment mice groups had a certain improvement,but the differences were not statistically significant(P>0.05).3.Cell experiment results:after high glucose(30mM)stimulated RAW264.7 macrophages,the expression of PKM2 protein was up-regulated(P<0.01),and the activation rate of M1 macrophage increased(P<0.05).After the treatment with 25ug/ml or 50ug/ml TFA,the expression of PKM2 protein was down-regulated(P<0.01),and the activation rate of M1 macrophage decreased(P<0.05).Conclusion:1.TFA significantly reduced the levels of proteinuria in db/db mice,reduced the renal pathological damage in db/db mice,reduced collagen fiber deposition,and inhibited the renal fibrosis in db/db mice,showing a dose-effectrelationship.2.TFA could inhibit local macrophage infiltration and M1 type macrophage activation in kidney tissue,reduce inflammation and fibrosis in kidney tissue,showing a dose-effect relationship;3.TFA inhibited the activation of M1 macrophages,which may be related to down-regulation of PKM2 protein expression. |
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