Mechanism Of Modified Dachaihu Decoction Regulating Mitophagy And Improving Insulin Resistance And Obesity Based On PINK1/Parkin Signaling Pathway

Obesity refers to chronic metabolic diseases caused by the interaction of environmental and genetic factors due to excessive fat accumulation and/or abnormal distribution and weight gain in the body.Insulin resistance(IR)is the link between both obesity and metabolism disorders.It is an important pathological mechanism of related complications caused by obesity,and the two often coexist and affect each other.In Traditional Chinese Medicine,it is believed that liver stagnation and spleen deficiency are the basic pathogenesis of insulin resistance and obesity,and Heat is an important pathological factor for its development.In combination with the pathogenesis of insulin resistance and obesity,we added classical drugs such as Fuling(Poria)and Baizhu(Atractylodes)on the original prescription of Dachaihu Decoction to strengthen Spleen and invigorate Qi,and named it "Modified Dachaihu Decoction(MDD)".In the previous experiments,it has also been proved that Modified Dachaihu Decoction,which has the effect of soothing Liver,strengthening Spleen and relieving Heat,can not only effectively reduce weight and improve insulin resistance,but also reduce oxidative stress and inflammation.Modern medical research has found that obese patients do have high levels of oxidative stress and low levels of chronic inflammation.Mitochondria are the main place for the production of reactive oxygen species(ROS),but they are extremely vulnerable to oxidative damage.Mitochondrial dysfunction is closely related to insulin resistance,while mitophagy is essential to clear damaged mitochondria and restore their function.Several other studies have shown that the deficiency of mitophagy is common in insulin resistance and obesity,and activating mitophagy can effectively alleviate insulin resistance and obesity.Therefore,in order to further explore the mechanism of MDD,we first established a mice model of insulin resistance and obesity with liver stagnation and spleen deficiency.Then the effects of MDD on mice of insulin resistance and obesity with liver stagnation and spleen deficiency were observed.Furthermore,the effects of MDD on mitophagy and mitochondrial function was further explored from the perspective of PINK1/Parkin signaling pathway.In this experiment,a mice model of insulin resistance and obesity with the syndrome of liver stagnation and spleen deficiency was firstly established by using the method of chronic constraint,clipping tail,excessive fatigue and daily high-fat diet.After successful modeling,the model was treated with MDD,and the positive drug was metformin.The general condition,weight,blood glucose and blood lipid of the mice were monitored during gavage.On the last day of the experiment,the elevated plus maze experiment was carried out and the levels of serum gastrin and D-xylose were measured to determine the improvement degree of liver stagnation and spleen deficiency.At the end of the experiment,serum ACTH and CORT levels were measured to determine the changes of the HPA axis,and serum insulin levels were measured and HOMA-IR was calculated to reflect insulin sensitivity.Meanwhile,the adrenal gland,spleen and thymus index were measured.At last,Part of the liver was taken out and used for HE staining to observe the pathological changes.Part of the liver was homogenized to measure the activity of antioxidant enzymes such as SOD,GSH-Px,the levels of oxidative stress products such as MDA,8-OHd G and inflammatory factors such as IL-6 and TNF-?.In addition,Real-time PCR and Western Blot were used to detect the expression levels of mitophagy-related proteins in the PINK1/Parkin signaling pathway.Finally,mitochondria of liver cells were extracted to measure mitochondrial membrane potential,mitochondrial swelling degree,ATP content,and the activity of key enzymes of the tricarboxylic acid cycle such as PDH and KGDH,to evaluate the changes in mitochondrial function.The results showed that a mice model of insulin resistance and obesity with liver stagnation and spleen deficiency could be established by chronic constraint,clipping tail,excessive fatigue and daily high-fat diet for 8 weeks.The characteristics of this model are as follows: There were significant symptoms of liver stagnation and spleen deficiency;Early short-term weight loss followed by increased weight and remained stable.The abnormality of blood lipid and hormone is similar to that of obesity.There was also immunosuppression and insulin resistance.And this modeling method is relatively simple and easy to operate.In further pharmacodynamic experiments,it was found that the administration of MDD had certain improvement on the above indicators to some extent,as shown in the following aspects: The general condition score and the level of serum gastrin and D-xylose were increased,while the body weight was decreased.In the elevated plus maze experiment,the proportion of open arm entry times and the proportion of open arm residence time increased.In terms of blood lipid,both TC and TG decreased while HDL increased.In terms of visceral index,adrenal index decreased and the index of spleen and thymus increased.In terms of hormone indicators,ACTH,CORT and insulin levels decreased,and HOMA-IR also decreased.In terms of oxidative stress and inflammatory mediators,the activities of the liver antioxidant enzymes such as SOD and GSH-Px increased,while the oxidative stress products and inflammatory factors such as MDA,8-OHd G,IL-6,TNF-? and CRP decreased significantly.At the same time,the expression of proteins related to the PINK1/Parkin mitophagy signaling pathway increased,and the mitochondrial function was restored.In summary,liver stagnation and spleen deficiency are important pathogenesis of both insulin resistance and obesity,and Heat plays a promoting role in the transformation of disease,and is an important pathological factor.Modified Dachaihu Decoction(MDD),which has the effect of soothing Liver,strengthening Spleen and relieving Heat,can not only alleviate the symptoms of liver stagnation and spleen deficiency,reduce body weight and blood lipid,but also can relieve hyperthyroidism of HPA axis and improve insulin resistance.It is speculated that the mechanism may be to activate the PINK1/Parkin signaling pathway and enhance mitophagy,so as to alleviate the damage of mitochondria caused by oxidative stress in the state of obesity and restore mitochondrial function.