Effect Of Modified Taohe Chengqi Decoction On Preventing Diabetic Macroangiopathy In Rats Based On AGEs-RAGE Pathway

Literature researchAs to the development of economy,changes of environment and living standard,as well as the acceleration of working rhythm and the aging of population,Diabetes mellitus had become one of the most important chronic non-communicable diseases of human being.Among all the diabetic complications,diabetic macroangiopathy,which contained coronary artery,cerebral artery and lower extremity arterial diseases,was the main complications that caused death and disability.The risk of macrovascular disease in patients with diabetes mellitus could be increased by 2-4 times compared with that of non diabetics,and 75%of patients with DM will eventually die of cardiovascular and cerebrovascular events induced by DM.Atherosclerosis was the main pathology of diabetic macroangiopathy.Study had confirmed that the early detection of AS,as well as early drug intervention,could delay the progression of AS,improve the patients’ prognosis and reduce the cardiovascular end point events.However,at present,western medicine was still lack of a sensitivity and specificity inspection index for early detection of AS.Moreover,drug treatment still had side effects such as bleeding,rhabdomyolysis.Thus,it was ergent to find a safer and more effective drug for clinical use.Although the pathogenesis of diabetic macroangiopathy were still complex,the excessive production of AGEs was an important pathogenesis of diabetic macroangiopathy.Many experimental and clinical studies also showed that serum AGEs had significant correlations with diabetic macroangiopathy,but there was a lack of evidence based evidence.There was no diabetic macroangiopathy in traditional Chinese medicine.Chest pain,heartache,dizzy,syncope,stroke,dementia,"pianku",Bi syndrome,sphacelus,fluid retention,blood stasis,Jue syndrome etc were the usual name related to diabetic macroangiopathy in TCM.Ancient and modern physicians had accumulated rich experience in the treatment of such diseases for thousands of years.Modified Taohe Chengqi decoction,a prescription based on Taohe Chengqi decoction from Shang Hanlun,was modified by professor Xiong Manqi from Guangzhou university of Chinese Medicine.Deficiency of Qi and Yin,blood and heat accumulation was the TCM,s pathogeny of ddiabetic macroangiopathy as to professor Xiong’ s experience.The former studies had showed that the Modified Taohe Chengqi decoction could lower blood glucose,regulating lipid metabolism,improve insulin resistance,protecting islet β cells,and finally improve the complications of DM.But as the pathogeny of diabetic macroangiopathy was complicated,professor Zhu believed that "Yang failing to promote health " was another key pathogeny of diabetic macroangiopathy,and Based on that theory,professor Zhu had changed some of the composition of professor Xiong’s Modified Taohe Chengqi decoction.Therefore,based on the AGE-RAGE signal pathway and oxidative stress,we tried to use animal experiment and,modern biological technology to figure out the possible mechanism of the three Modified Taohe Chengqi decoction for treating diabetic macroangiopathy and optimize the composition of Modified Taohe Chengqi decoction.Evidence based researchObjectiveThe excessive production of AGEs was an important pathogenesis of diabetic macroangiopathy.Current studied had found that there were significant differences between serum AGEs levels of diabetic macroangiopathy patients and patients with only diabetic mellitus,as well as patients with only diabetic mellitus and healthy people.But there were still no exact conclusion yet.Therefore,this study intended to use Meta analysis,through searching database of home and abroad,to explore the correlation of between AGEs levels and diabetic macroangiopathy,and so as to provide evidence for the clinical application of AGEs levels.MethodsAccording to the initial inclusion and exclusion criteria,we searched databases at home and abroad about studies contained of AGEs levels,diabetic macroangiopathy patients,patients with only diabetic mellitus and healthy people.After selecting the included studies,we extracted datas and use RevMan 5.3 to perform a Meta analysis.Finally,we analyzed the results of Meta analysis about the correlation between AGEs levels and diabetic macroangiopathy,and use sensitivity analysis to handle the heterogeneity.Funnel plot was used to evaluate the publication bias.ResultsA total of 14 studies and 1290 patients were included.1290 patient included 338 cases of diabetic macroangiopathy,607 cases of patients with only diabetic mellitus and 345 cases of healthy people.The results of Meta analysis showed that there were significant differences about AGEs-CML levels between diabetic macroangiopathy patients and patients with only diabetic mellitus,as well as patients with only diabetic mellitus and healthy people(P<0.01).And the results were still stable after the sensitivity analysis.When it come to levels of AGEs,there were significant differences between patients with only and diabetic mellitus and healthy people(P<0.01),and after a sensitivity analysis,there were significant differences between diabetic macroangiopathy patients and patients with only diabetic mellitus(P<0.01).ConclusionAs AGEs-CML was a kind of represented AGEs,there were significant differences between diabetic macroangiopathy patients and patients with only diabetic mellitus.One of the possible effect might be the excessive production of AGEs.The Meta analysis concluded that AGEs levels could increase specially in diabetic macroangiopathy patients.However,as to the existed of low qualities researches and heterogeneity,it still needed further researches to evaluate the possibility for AGEs to diagnosis diabetic macroangiopathy.Experimental researchObjectiveBased on the experience of Professor Zhu Zhangzhi,he changed some of the composition of professor Xiong’ s Modified Taohe Chengqi decoction.Then we followed such changes and set TCM group 1(professor Xiong’ s original Modified Taohe Chengqi decoction),TCM group 2,TCM group 3,and western medicine group.Secondly,based on AGE-RAGE pathway and oxidative stress,we used animal experiments,HE staining,QRT-PCR,immunohistochemistry,electron microscope,Western blotting,ELISA and other methods to explore the possible mechanism and better compatibility of Modified Taohe Chengqi decoction on treating diabetic macroangiopathy.Methods120 male SD rats were firstly fed for 1 weeks,then 10 of them were selected into the normal group,which were fed by normal diet.The rest were selected as model group,which were fed with high-fat diet for 6 weeks.After 6 weeks,we made pre experiments,and then found out 40 mg/kg dose of STZ was the best dose.Thus we injected the rest 89 rats with 40 mg/kg dose of STZ while the normal group rats were injected with equal amount of citric acid.After twice of FPG detection,FPG stability above 11.1mmol/L was established as T2DM model.Finally,74 T2DM model rats were successfully made.Then the 74 T2DM model rats were randomly divided into T2DM model group for 14 rats,AG group for 15 rats,TCM group 1 for 14 rats,TCM group 2 for 15 rats and TCM group 3 for 15 rats.The total intervening period were 8 weeks.We checked the general situation such as weight,food intake,water intake and FPG every 4 weeks.At the end of the experiment,Fins,HOMA IR,HBA1C,TC,TG,HDL-C,LDL-C,SOD,CAT,GSH-Px,etc were measured.Aorta pathology morphology were observed by light microscopy and electron microscope.AGEs were measured by IHC and elisa.Western blotting was used to detect ERK1/2 protein expression.RTQ-PCR was to detect the mRNA expression of RAGE mRNA、eNOS mRNA、p47phox mRNA.Results1.General situation:the normal group rats were in good mental condition,tender and smooth skin,shiny hair,stable weight and urine volume,no more water intake,no more food intake.The rats in the model group showed obvious symptoms of polydipsia,polyuria,polyuria,emaciation,poor spirit,slow reaction,dry hair,dry skin,and a small amount of feces.After drug intervention,the rats in 4 drug intervention group were improved in mental state,physical signs,body weight and symptoms,but the difference was still worse than those in the normal group.Finally,a total of 11 rats died,in which the model group died 4 of the rats,TCM group 1 died 3 of the rats,TCM group 2 died 2 of the rats,TCM group 1 died 1 of the rats,AG group died 1 of the rats.The main causes of death were considered as DKA,HHS,and gastrointestinal complications.At the end of the experiment,a total of 71 rats were completed,including the normal group(n = 10),the model group(n=10),the TCM group 1(n = 11),the TCM group 2(n = 12),the TCM group 3(n=14)and the western medicine group(n = 14).As to body weight,food intake,water intake,weight of normal group increased steadily with time migration,water intake and food intake did not change significantly;the weight of the model group decreased,and water intake,food intake increased gradually;the analysis on interaction between time factors,intervention factors,time factors and intervention factors,each drug intervention group were statistically significant(P<0.01);after 8 weeks of drug intervention,each drug intervention group was significantly better than the model group(P<0.01),but still less than the normal group(P<0.01)in increasing body weight,reducing water intake,food intake.Among the 4 drug intervention groups,the TCM group 3 was significantly better than other group in increasing body weight,reducing water intake(P<0.01),but there were no significant differences between other drug intervention groups in food intake(P>0.05).2、Glucose metabolism:after 8 weeks of drug intervention,the 4 drug intervention groups were significantly lower than the model group in FPG and HbAlc(P<0.01),but still significantly higher than normal group(P<0.01),and TCM group 1,TCM group 2,AG group were significantly higher than TCM group 3(P<0.01).As to Lipid metabolism,after 8 weeks of drug intervention,there were no significant differences between the drug intervention groups in TC,TG,LDL-C,HDL-C;the 3 TCM groups were significantly decreased when compared with the model group in TC(P<0.01),but there was no significant difference between the AG group and model group(P>0.05).In TG,LDL-C,HDL-C the 3 TCM groups were significantly lower than the model group(P<0.01),but there was no significant difference between the AG group and model group(P>0.05).As to IR,the 3 TCM groups were significantly lower then the model group(P<0.01),but still significantly higher then normal group(P<0.01),and there was no significant difference among each drug intervention group(P<0.01).3.AGEs-RAGE pathway:after 8 weeks of drug intervention,each control groups had significantly increased AGEs levels compared with the normal group(P<0.01).And the model group mostly increased,the TCM group 1 showed no significant difference than TCM group 2,AG group and TCM group 3 were significantly decreased than TCM group 1,TCM group 2(P<0.01),TCM group 3 showed no significant difference than AG group.As to changes in IHC IOD value,it was similar with(P>0.05)the AGEs levels.Image showed that AGEs showed brown particles precipitate in the thoracic aorta of rats in each group,normal group showed sparse brown particles precipitate;model group showed large amount of brown particles precipitate,deep dyeing in endothelial and smooth muscle layer,vascular wall interstitial.The 4 drug intervention group showed more brown staining precipitation than the model group,but lighter than the normal group.The TCM group 3 and AG group showed lighter compared with TCM group 1 and TCM group 2.TCM group 3,which pale staining,brown granules mainly deposited on the endothelial layer,smooth muscle layer were sparse and lightly stained brown granules;TCM group 1,TCM group 2,AG group showed increased deep dyeing brown yellow particles precipitate in endothelial and smooth muscle layer.As to RAGE mRNA,the 4 drug intervention group decreased significantly than model group(P<0.01),TCM group 1 showed no significant difference with TCM group 2(P>0.05),AG group decreased significantly than TCM group 1(P<0.01),TCM group 2,TCM group 3 showed no significant difference than AG group(P>0.05),4.Oxidative stress:the changes of SOD,CAT and GSH-Px in each group were similar.The normal group increased significantly than the other groups(P<0.01),the 4 drug intervention groups increased significantly than the model group(P<0.01),TCM group 3 increased significantly than the other drug intervention groups(P<0.01),TCM group 1 and TCM group 2 showed no significant difference than AG group(P>0.05).As to P47phox mRNA,after 8 weeks of intervention,model group and drug intervention groups was significantly higher than the normal group(P<0.01),TCM group 1 decreased than the model group(P<0.05),TCM group 2,TCM group 3,AG group were significantly decreased than model group(P<0.01),TCM group 3 decreased significantly than the other drug intervention groups(P<0.01).5、The endothelial function showed that the expression of mRNA in eNOS,the normal group increased significantly than the model group and drug intervention groups(P<0.01),the model group significantly decreased than the drug intervention group(P<0.01),TCM group 3 increased significantly than the other drug intervention groups(P<0.01);there were no significant differences among TCM group 1,TCM group 2 and AG group(P<0.05).In the expression of ERK1/2,the change trend between each group were similar.The result of WB showed that the normal group decreased significantly than model group and drug intervention groups(P<0.01);the intervention group were significantly lower than model group(P<0.01);The 3 TCM groups were significantly lower than AG group(P<0.01);there was no significant difference among the TCM groups(P>0.05).6.As to HE staining,the normal group showed that the endothelial cells were closely linked,no shedding,no swelling degeneration,no foam cell formation,smooth muscle layer were also closely linked,no abnormal proliferation.The model group showed that endothelial cell layer shedding serious,cell swelling significantly,visible foam cells and macrophages,smooth muscle layer structural disorder,obvious proliferation of smooth muscle cells,endothelial layer formed in the fibrous cap;The 4 drug intervention groups were reduced compared with model group;TCM group 1 showed endothelial cell loss,swelling,a small amount of endothelial layer lipid deposition,foam cell formation,smooth muscle cell link disorder,abnormal proliferation of cells,but no fibrous cap formation;TCM group 2 showed endothelial cells swelling,lesion degree than TCM group 1,no obvious foam cells,fibrous cap formation,smooth muscle layer visible abnormal cell proliferation,cell link not the rule;TCM group 3 showed endothelial cells swell slightly,but still close links between the cells,no foam cells,fibrous cap formation,disease history compared to other drug intervention groups,the smooth muscle layer had no obvious abnormal proliferation of endothelial cells;AG group showed slightly swelling,a small amount of foam cell formation,no fibrous cap formation,smooth muscle cells arranged in disorder,loose.ConclusionIn improving the general condition,Modified Taohe Chengqi decoction could effectively reduce water intake,food intake,and effectively increase the body weight of rats;As to blood glucose,Modified Taohe Chengqi decoction could improve insulin resistance,reduce FPG and HbAlc play a stable hypoglycemic effect;As to blood lipid,Modified Taohe Chengqi decoction could improve lipid metabolism effect by reducing TG,TC,LDL-C,raising HDL-C;As to the protection of vascular disease,Modified Taohe Chengqi decoction could through the AGE-RAGE pathway,reduce the formation of AGEs,reduce the expression of RAGE receptors,thereby inhibiting the AGE-RAGE pathway,and increase serum SOD,CAT and GSH-Px levels,decreased p47phox mRNA expression,to repair the antioxidant ability.Finally,by increasing eNOS mRNA,decreasing the expression of ERK1/2 protein,Modified Taohe Chengqi decoction had an effect on improving endothelial function,reducing abnormal increased vascular smooth muscle cells,as well as slowing down the progression of atherosclerosis.In addition,the experiment also found that although the aminoguanidine could effectively reduce the formation of AGEs,but its atherosclerosis effect was weak,which accounted that the mechanism of diabetic macroangiopathy were still many targets.Large sample,multi center and further researches were still needed to confirmed.On the other hand,comparison between groups showed that compared to other drug intervention groups,TCM group 3 could be more effective in inhibiting the progression of AGE-RAGE pathway,oxidative stress and slow down the progression of atherosclerosis.As to the TCM theory,TCM group 3 had strengthened the "Wen Yun Yang" role,which showed that diabetic macroangiopathy also had other characteristics liked Yang failing to promote health besides Deficiency of Qi and Yin,blood and heat accumulation.The above suggested that the future of TCM treatment for diabetic macroangiopathy should also focus on "Wen Yun Yang" besides previous Nourishing Yin and Qi and activating blood.But all in all,it needed further clinical and experimental studies to confirmed.