Huangqi Guizhi Wuwu Decoction Protects Diabetic Vascular Injury Through ARG1-NO Pathway

ObjectiveDiabetes mellitus is a comprehensive metabolic disease characterized by continuous elevation of blood glucose,which clinical manifestations including polydipsia,polyuria,polyphagia,emaciation,fatigue,and obesity etc.Long-term hyperglycemia will lead to the occurrence and development of a variety of complications,among which the most common is diabetic vascular complications,and the main pathological manifestation is diabetic vascular injury.However,the etiology of diabetic vascular injury is unknown,and there is currently a lack of effective clinical treatment,and it is an urgent clinical problem to find safe and effective drugs.It is a feasible strategy to find the corresponding method from the classical prescription to solve the refractory diseases such as diabetic angiopathy.In this study,C57BL/6J diabetic mice model induced by abdominal cavity injection of streptozocin(STZ)were used to explore the effect and mechanism of Huangqi Guizhi Wuwu Decoction(HGWWD)on diabetic vascular disease.Methods1.Using triple quadrupole time-of-flight mass spectrometry technique(HPLC-QTOF-MS/MS),Tested the 6 batches of different time of HGWWD freeze-dried powder samples and determined HGWWD mainly 5 chemical composition of astragalus glycosides,calycosin separately,glucoside,cinnamic aldehyde,paeoniflorin,6-gingerol,to determine HGWWD chemical composition and the accuracy and repeatability of monitoring results of HGWWD.2.Using STZ(50 mg/kg/d)five days continuous intraperitoneal injection preparation diabetes(diabetes mellitus,DM)mouse model to induce diabetic vascular complications.3.According to fasting blood glucose and body weight,mice were randomly divided into 6 groups:control group(Cont.),diabetes model group(DM,purified water,ig.),HGWWD low-dose group(DM+L-HGWWD,30 g/kg/d,ig.),HGWWD medium-dose group(DM+M-HGWWD,60 g/kg/d,ig.),HGWWD high-dose group(DM+H-HGWWD,120 g/kg/d,ig.)and HGWWD medium-dose+L-NAME group(DM+M-HGWWD+LNAME).4.The reactive congestion time and microcirculation blood perfusion volume in the lower extremity of mice in each group were measured by PERIMED PSI System.5.The blood flow velocity(MV)and vascular resistance index(PI)of the left femoral artery of mice in each group were detected by Vevo 2100 Ultrasound Imaging System.6.The vasoconstriction and diastolic function of mice was detected by Multi Myograph System DMT620.7.According to the principle of nitrate reductase,the content of serum nitric oxide(NO)was determined by total nitric oxide detection kit.8.NO levels in blood vessels were detected by Daf-2DA fluorescence staining.9.The arginase activity of aorta in each group was evaluated by measuring the urea level produced by L-arginine.10.Quantitative reverse transcriptionPCR(Q-PCR)was used to detect mRNA levels of ARG1 and ARG2 in aorta.11.using Western blot(WB)to determine the protein expression of ARG1 in aorta.12.The aorta was incubated with drug-containing serum in vitro,and the effect of ARG1 overexpression on HGWWD efficacy was measured using adenovirus overexpression of ARG1.13.Mice with endothelial-specific ARG1 knockout were used for STZ modeling and administration to determine the effect of endothelial-deficient ARG1 on the efficacy of HGWWD.The group was divided into 5 groups:Cont.+A1 loxp/loxp group,DM+A1 loxp/loxp group,DM++A1 A1loxp/loxp +HGWWD group,DM+EC-A1-/-group,DM+EC-A1-/+HGWWD group.Results1.According to the result of HGWWD total component analysis study,HGWWD was detected in 31 chemical elements by HPLC-Q-TOF-MS/MS,in which there are 11 chemical composition belongs to Astragalusmembranaceus(huangqi),seven chemical constituents from cinnamon twig(guizhi),7 chemical composition of belonging in Radix Paeoniae Alba(baishao),3 chemical constituents from ginger(shengjiang),3 chemical components belonging to jujube(dazao);At the same time,the detection results of HGWWD are accurate,reliable and repeatable;2.STZ-induced diabetic mice showed delayed weight gain or weight loss and blood sugar level consistently higher than 250 mg/dL(or 13.8mmol/L);3.Compared with the normal group,DM+M-HGWWD group(60 g/kg/d,ig.)showed significantly increased terminal microcirculatory blood perfusion after 2 weeks of administration(P<0.05),thus confirming that the optimal dose of HGWWD was 60 g/kg/d for 2 weeks of administration;4.From 6 weeks of STZ-induced diabetes,HGWWD treatment was given for 2 weeks.Compared with the DM group,the MV of the left femoral artery was significantly increased in the HGWWD treatment group,and the PI was decreased,but the body weight and blood glucose of diabetic mice were not changed.5.Compared with normal group,DM model mice showed significantly longer reactive hyperemia time in terminal microcirculation of the lower extremities after 8 weeks of modeling(P<0.05).vascular endothelial dependent contraction diastole function(P<0.05)decreased,blood vessel and serum NO levels(P<0.05)reduced,and vascular ARG activity increased(P<0.05).HGWWD(60 g/kg/d,ig.)treatment could significantly reverse the above pathological indexes after two weeks of administration(P<0.05),L-NAME administration for two weeks could reverse the improvement effect of HGWWD on endothelium-dependent vasodilation.6.ARG1 mRNA and protein levels were significantly increased(P<0.05)in DM mice,two weeks of HGWWD could reduce ARG1 mRNA and protein expression levels significantly.7.Compared with HG+HGWWD group,ARG1 overexpression could significantly reduce the protective effect of HGWWD drug-containing serum on endothelium-dependent aortic vasodilation induced by high glucose(P<0.05)..8.Compared with DM++A1 loxp/loxp +HGWWD group,DM endothelial-specific ARG1 knockdown did not further increase the efficacy of HGWWD administration group(P>0.05).Conclusion1.Thirty-one chemical components of the lyophilized powder of HGWWD were identified by HPLC-Q-TOF-MS/MS,which realized a more comprehensive analysis of the compound components of HGWWD,and provided a new scientific basis for the quality control of HGWWD.2.HGWWD administration can effectively improve STZ-induced vascular injury,Meanwhile,it suggests that HGWWD treatment may protect vascular endothelial dysfunction through the regulation of endothelial ARG1-NO signaling pathway in diabetic model.