Study Of The Protective Effect And Mechanism Of BWD On Type Ⅰ Diabetes Complicated With Liver Injury Based On Network Pharmacology

Objective Network pharmacology and type 1 diabetes mellitus（T1DM）mouse model were used to analyze the protective effects and the underlying mechanisms of Baihe Wuyao Decoction（BWD）on diabetes associated hepatic injury.Methods 1 Firstly,network pharmacology was used to analyze the effective chemical components of BWD,and its related targets and signal pathways were screened.2Auto Dock software was used to verify the molecular docking of the potential active components of BWD with one key target proteins:Akt.3 After the Network pharmacology analysis,75 SPF male healthy Kunming mice aged 6～8 weeks were divided into control group（CNTR）,model group（Model）,positive group（INS）,high-dose（HD）,medium-dose（MD）,low 1 dose（L1D）,and low 2 dose（L2D）of BWD groups.The CNTR group was injected with citrate buffer,and the other groups were given STZ solution(10 m L·kg^-1).After 5 days,fasting for 6 hours,blood glucose was collected from the tail tip,and the mice with blood glucose higher than 16.7 mmol·L^-1were regarded as T1DM being developed.The mice in INS group were injected with insulin(0.091 U·kg^-1)subcutaneously twice a day.The mice in HD,MD,L1D and L2D groups were intragastrically given with 15,5,2.5 and 1.25 g·kg^-1·d^-1,respectively,for continuous 6weeks.Then the mice were fasted for 12 hours,and were anesthetized with isoflurane.Then the blood was taken from the abdominal aorta and the liver samples were collected.An automatic biochemical detector was used to detect the content of serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）.The liver pathological changes were observed by HE staining.The antioxidant capacity of liver tissue was evaluated by the content of superoxidedismutase（SOD）and malondialdehyde（MDA）in liver homogenate.The liver protein level expression of p-AKT and AKT by Western blot.Quantitative real time polymerase chain reaction（Q-PCR）method to detect manganese superoxide dismutase（Mn-SOD）,nitric oxide synthase（i NOS）,inflammatory factors include tumor necrosis factor（TNF-α）,nuclear factor-κB（NF-κB）,interleukin-1β（IL-1β）,interleukin-6（IL-6）,interleukin-8（IL-8）,apoptosis factors include B-cell lymphoma-2（Bcl2）,Bcl2-associated X protein（Bax）,Caspase 3（CASP3）and cell proliferation factors activator protein-1（AP-1）at the m RNA level.Results 1 The results of Network pharmacology showed taht:（1）after database analysis,a total of 20 active components from BWD,589 predicted targets and 111 common targets related to T1DM and associated hepatic injury were obtained.The core target molecules mainly included AKT1,MAPK3 and so on.（2）GO functional enrichment analysis involved biological processes including positive regulation of protein serine/threonine kinase activity,positive regulation of MAP kinase activity and so on.Membrane raft,membrane microdomain and so on involved in the expression of cellular components.The processes related to molecular functions mainly included transmembrane receptor protein kinase activity and transmembrane receptor protein tyrosine kinase activity.（3）KEGG pathway enrichment analysis involved AGE-RAGE signaling pathway in diabetic,Foxo signaling pathway and so on.（4）"Chinese medicine-ingredients-targets-pathways"constituted an integrated network of multiple components,multiple targets,and multiple pathways.（5）the results of molecular docking showed that the core components of BWD have good binding activity with the targets,including quercetin,norboldine,beta-sitosterol and stigmasterol,which verified the accuracy of network construction prediction.2Compared with the CNTR,the detection results of Model can undergo the following changes:（1）the concentration of ALT and AST in the serum increased,and the relative weight of the liver increased.HE staining showed that the liver cells were swollen and loosely arranged.（2）the blood glucose level was abnormally increased,indicating abnormal liver glucose metabolism in mice.（3）the expression of MDA and i NOS increased,the expression of Mn-SOD,SOD decreased,and the ability of mouse liver to scavenge free radicals was weakened.（4）Western blot experiment results showed that the expression of p-AKT/AKT decreased indicated that the insulin signaling pathway is abnormal.（5）Q-PCR results showed that the increased expression of TNF-α,NF-κB,IL-1β,IL-6,and IL-8,which indicated that STZ stimulation caused liver inflammation in mice.（6）the expressions of Bcl2/Bax and AP-1 decreased,and the expression of CASP3increased,which infered that there was apoptosis in the liver of mice.3 Compared with the Model,BWD test results can undergo the following changes:（1）the content of ALT and AST in serum has been reduced,and the pathological changes of liver tissue have been improved.（2）blood glucose levels were improved.（3）the expression of MDA and i NOS was down-regulated,and the expression of Mn-SOD and SOD increased.The antioxidant capacity of the mouse liver was enhanced to protect the liver cell membrane from damage.（4）the expression of p-AKT/AKT was up-regulated,and the insulin signaling pathway was improved.（5）the expression of TNF-α,NF-κB,IL-1β,IL-6,and IL-8 decreased,and the liver inflammation in mice were reduced.（6）the expression of Bcl2/Bax and AP-1increased,the expression of CASP3 decreased,and the liver cell apoptosis of mice were reduced.Conclusion BWD could play a protective effect on diabetes complicated liver injury through regulating the activity of AKT in the liver,anti-oxidation,anti-inflammatory,anti-apoptosis and regulating cell proliferation.Figure 18;Table 6;Reference 144...