Study On Protective Effect And Mechanism Of Shexiang Tongxin Dripping Pill In Lower Limb Ischemia Injury In Diabetic Mice

Peripheral artery disease(PAD)is one of the common great vessel complications aroused by Type 2 diabetes mellitus(T2DM).It often involves the tibial and peroneal arteries of the lower extremities and induces the distal limb ischemia,resulting in the occurrence of diabetic foot complications,which is closely related to the disability and death of diabetic patients.Diabetic PAD usually starts latently and lacks typical symptoms.When intermittent claudication,limb pain and skin ulcers occur,the lumen stenosis of the lower extremity artery is usually more than 50%.Therefore,it is very important to identify early by measuring ABI(ankle-brachial index)and color Doppler ultrasound.Angiography is the "gold standard" for the diagnosis of PAD,which can help doctors formulate feasible revascularization strategies.The treatment of diabetic PAD includes risk factor control,hypoglycemic,ester regulating,antiplatelet and vasodilator drugs,and vascular revascularization.For patients with severe lower extremity ischemia,the rate of limb salvage can be up to 90% after surgical or endovascular treatment.In addition,gene and stem cell therapy as a new therapeutic method can provide new ideas for prevention and treatment of diabetes mellitus by promoting angiogenesis and vascular endothelial repair.However,there is still no large sample and high quality randomized controlled trials proved its long-term efficacy and adverse reactions.The angiogenesis ability of diabetic PAD patients is obviously insufficient.Some factors inhibit angiogenesis and arterial production included hyperglycemia tissue microenvironment,the weakening of vascular endothelial growth factor(VEGF)response and the decrease of circulating endothelial progenitor cells mobilization ability.At present,gene and stem cell therapy is in pre-clinical research.Molecular targets for therapeutic angiogenesis in the future remain the focus of PAD research.Chinese medicine has a long history of treating atherosclerotic cardiovascular disease and diabetes.In recent years,domestic scholars are committed to exploring the mechanism of traditional Chinese medicine to protect the function of vascular endothelial cells and promote angiogenesis,so as to fill the gap of traditional Chinese medicine pharmacology.Previous studies have shown that many Chinese herbal compounds or monomers can reduce oxidative stress and improve diabetic vascular injury,thereby promoting angiogenesis and improving endothelial function for alleviating tissue and organ ischemia.Our laboratory is committed to the experimental study of Shexiang Tongxin dripping pill(STDP).STDP is composed of seven traditional Chinese medicines.It has the effects of Nourishing Qi,dredging pulse,promoting blood circulation,removing blood stasis and relieving pain.In our previous experimental study on improving microcirculation disorder of coronary heart disease,we found that the mechanism of STDP is multi-target and multi-channel,which can reduce vascular endothelial cell damage and inflammatory response,and oxidative stress,accelerating microcirculation blood flow velocity and so on.In addition,domestic scholars explored the mechanism of STDP based on serum pharmacochemistry and network pharmacology,and also found that STDP can activate PPAR-γ that can regulate glucose metabolism and improve insulin resistance.Therefore,STDP was as an observation drug,taking into account both the commonness of atherosclerotic macrovascular diseases and the characteristics of diabetes itself.In this study,we constructed a diabetic mouse model of lower limb ischemia to observe the improvement effect of Shexiang Tongxin dripping pills on lower limb ischemia and explore its mechanism,so as to find new targets for the treatment of diabetic PAD.Objective Establish a hindlimb ischemia model by femoral artery ligation with db/db diabetic mouse,to observe the protective effect of STDP on hind limb ischemic injury and to explore its possible mechanism.Method 1.A total of 15 10-12 weeks old male db/db diabetic mice,weighing 40±5g,were selected.The left lower limb was selected,a hindlimb ischemia model was constructed by femoral artery ligation and transection method.The blood flow of lower limbs was tested after 30 minutes operation by laser doppler.The blood flow of the ischemia side decreased to less than 50% of normal side were considered to be successful models.2.Randomly divided mice into 3 groups,pseudo-surgery group(Sham group,n=5),model group with normal saline group(MOD+NS group,n=5)and model with STDP group(MOD+STDP group,n=5).After surgery,the model group was continuously given NS or STDP via gavage for 2 weeks.3.All mice took laser scan on the d3,d7 and d14 respectively to detect blood flow on both lower limbs to observe the recovery difference,ischemia / ischemic lower limb blood flow ratio was calculated and analyzed.The symptoms of ischemic injury in the lower extremities were assessed by lower limb activity scale(1～6 points).At d14,all animals were executed;separate the lower limb muscle tissue for HE staining,CD31 histochemical stain and immunofluorescence stain,ELISA for VEGF,Ang-1,Tie-2,PDGF expression,Western blot for VEGFR and Ang-1 protein expression.Results 1)Mice in STDP group achieved 6 points of activity assessment at the end point,on d14 mice in STDP group basically restored limb function to the preoperative level that surgical hindlimb can bear body weight without movement dysfunction while those in NS group had 5 for median,activity recovery was low,at the endpoint some mice got normal hindlimb movement.2)Laser scan of ischemia/non-ischemia limb ratio was about 0.39 on average after modeling,blood flow decreased more than 50%,model succussed.Blood flow improvement was more obvious and significant in STDP group compared to NS group,with an average ischemia/non-ischemia ratio of 0.79,0.90 on day 7 and d14 respectively,while that of NS groups was 0.59,0.65 respectively,at the endpoint,there is obvious difference between blood flow ratio(p<0.01).3)On HE staining there was tissue loosen,myofiber histolysis and rupture in NS group section,while STDP group showed more normal muscle tissue with anatomical structure in order and no myofiber atrophy.CD31-positive cells in hindlimb section of STDP group was significant increased than that of NS group(p<0.01),furthermore,on immunofluorescence staining,there were more CD31-positive of STDP group(p<0.0001).4)ELISA test showed more CRCX4 factors in hindlimb of STDP group(p<0.05),while no obvious increase of VEGF,PDGF,Ang-1、Tie-2;in WB test there were no obvious change on VEGFR and Ang-1 protein expression.Conclusion STDP can promote the repair of ischemic injury of the lower limb in diabetic mice,improve the blood perfusion of the lower limb,and protect the structure and function of the ischemic tissue.We speculate that this protective effect is related to protecting endothelial cell function and promoting arteriogenesis.