Effect Of Artesunate On Cardiovascular Complications In Periodontitis With Type Ⅰ Diabetes Rat Model And Related Mechanisms

Objective:To explore the possible therapeutic potential of artesunate for the protection against cardiovascular complications from type I diabetes and periodontal disease with type I diabetes rats and possible underlying mechanisms.We also aim at the relevant effects of oral and vascular flora changes.Method: Part Ⅰ: Type Ⅰ diabetes rat model was utilized to measure the protective effect of artesunate(50,100 mg/kg)on cardiovascular.Blood samples were collected to determine blood glucose and blood lipids related biochemical indicators change.Myocardium and aortic arch were stain with HE and Masson for observing pathological changes.RT-PCR and immunohistochemistry assays were used to examine the expression of RAGE,NF-κB,MMP9,MMP1 and CD68 in the cardiovascular.PART II: Construct rat models of Type I diabetes(DM),periodontitis(PD)and type I diabetes with periodontitis(DM+PD),rat models were established.The(DM+PD)rats were intervened with(10,30,60 mg/kg)artesunate respectively through intragastric administration.Health rats,PD,DM,DM+PD groups were set up as untreated control groups.Body weight and fasting blood glucose of rats were measured.Oral swabs were used to collect oral flora and determine the changes of oral flora in each group.Blood samples were collected to determine changes in blood glucose,total cholesterol,low-density lipoprotein cholesterol,high-density protein cholesterol and C-reactive protein;Mandibular molars,alveolar bone,heart and aortic arch were taken for imaging examination and micromorphological analysis.Micro-CT was used to observe the changes of alveolar bone structure and bone density of the mandibular molar region.Rat hearts were weighed and calculate the heart index,and heart slices were stained with Masson,Sirius red and Tunnel staining solution for observing myocardial fibrosis and apoptosis.Separate the aortic arch tissue of rats for determining changes of intravascular microflora and the relationship with oral micro flora.Immunohistochemical assay,RT-PCR were used to examine the expression of NF-κB,TLR4 and VEGF in alveolar bone tissue and expression of NF-κB,TLR4,VEGF,ICAM-1,p38 MAPK,TGF-β,Smad2 and MMP9 in cardiovascular tissue.Result: Part Ⅰ: The results suggest that heart weight and body weight of diabetic group are decreased,the heart-to-body ratio was slightly increased and blood glucose and blood lipids were significantly increased(P<0.05).Treatment of artesunate significantly improved the condition of diabetes,DM+ ART(100mg/kg)group has a significant effect on blood glucose decline(P<0.05),and M+ART(50 mg/kg)group significantly improves heart weight and blood lipid sublimation indicators(P<0.05).H&E and Masson staining results suggest that50 mg/kg artesunate treatment has a recovery effect on tissue morphological abnormalities and fibrosis.Immunohistochemistry and RT-PCR results suggest that treatment with artesunate decreased the expression of NF-κB,CD68,MMP1,MMP9 and RAGE(P<0.05).The expression of RAGE protein in cardiovascular tissue was more significantly reduced by 100 mg/kg artesunate treatment(P<0.05).Part Ⅱ: The results suggest that the biochemical indicators of the periodontitis group are close to the control group,there is no significant different.In type I diabetes and type I diabetes with periodontitis rat model,body weight and heart weight were reduced and blood glucose and blood lipid were significantly increased(P<0.05).Treatment with 60 mg/kg artesunate can significantly improve the situation(P<0.05).Micro-CT result showed that treatment with 60 mg/kg artesunate can effectively improve alveolar bone resorption and bone density reduction(P<0.05).Staining assays suggest that treatment of 60 mg/kg artesunate has a significant therapeutic effect on myocardial apoptotic fibrosis(P<0.05).High expression of NF-κB,TLR4,VEGF,ICAM-1,p38 MAPK,TGF-β,Smad2 and MMP9 in the alveolar bone and cardiovascular tissue in type I diabetes and type I diabetes with periodontitis rat model were reduced after treatment of artesunate with a concentration dependent effect(P<0.05).Sequencing result suggest that rats in each model group had dysbiosis in the vascular flora and oral flora.Treatment with artesunate can adjust the dysbacteriosis,60 mg/kg artesunate treatment can significantly improve the composition of intravascular flora by reducing the proportion of Proteus and increasing the composition of thick-walled bacteria;10 mg/kg artesunate treatment can effectively improve oral microflora composition.Conclusion:1.Artesunate have a protective role against hyperglycemia and cardiovascular complications in diabetic rats by inhibiting the expression of proteins in the RAGE/NF-κB signaling pathway and downstream inflammatory factors.2.Periodontitis related pathogenic bacteria cause dysbiosis of oral and intravascular flora of type I diabetes and aggravate cardiovascular complications via the oral-vascular pathway.3.Periodontitis can promote occurrence of cardiovascular complications of type I diabetes.The possible mechanism is that the NF-κB/p38 MAPK/TGF-βcascade pathway induced myocardial apoptosis fibrosis,and the NF-κB/TLR4 pathway causes inflammation changes of large blood vessels.4.Artesunate can effectively improve the progression of periodontitis and cardiovascular complications of type I diabetes by inhibiting apoptosis,inflammatory pathways and its downstream factors in a concentration-dependent manner.