The Influence Of PI3K/Akt/AP-1Pathway On The Proliferation Of Smooth Muscle Cells In High Glucose And The Expression Of CRP, IL-6, TNF-α

Objective: Diabetic vascular complication is characteristic of high speedof development, multiple positions, and atherosclerosis and it is primary causeto death of diabetic patients.Vascular smooth muscle cells are the major cellsin the constitution of vessels. The proliferation of VSMCs can cause thereconstruction of vessels, narrowing of the lumen and dysfunction ofvasomotor regulation, which is the major pathologic change of atherosclerosis.Many scholar think both diabetes and atherosclerosis are highly related to lowlevel of chronic inflammation state.In the pathogenesis of inflammation ofdiabetes and atherosclerosis, inflammatory cytokine is considered to be themajor hazards factor in causing the morbidity of these two diseases andaffecting the diseases evolution. Study shows that as inflammatory indicator,CRP, IL-6, TNF-α are involved in the pathological process of the vasculopathy.Activator protein-1participates in the regulation of inflammatory cytokine inthe construction of vessels, which can play an important role in cellproliferation and inflammatory reactions. It is also showed that AP-1can haveimportant regulation effect on inflammatory factor of atherosclerosis in theformation of atherosclerosis and then regulate and control the formation anddevelopment of atherosclerosis.Phosphatidylinositol-3-kinases signal pathway and its main downstreameffector protein kinase B (PKB/Akt) take part in the differentiation,proliferation, apoptosis and migration of cells. They can regulate the AP-1transcription factors to couple on specific starter or to promotor to increase theactivity of inflammatory medium gene, which finally result in the generationof many cell factors by cells. Therefore, PI3K/Akt signal pathway plays a vitalfunction in the regulation process of inflammatory response reaction of bodies. LY294002hinders the activation of PI3K through targeted inhibition ofcatalytic subunit p110of PI3K and then restrain the activation of itsdownstream targeted moleculars PDK and Akt, which makes it become themost classical inhibitor in the study of PI3K/Akt.Currently, however, the studies about the signal expression ofPI3K/Akt/AP-1pathway in vascular smooth muscle cells and the role ofPI3K/Akt pathway in VSMCs proliferation and inflammatory caused by highglucose are rare. Therefore, the key point of this work is to study the VSMSsproliferation and the expression mechanism of inflammatory factors CRP,IL-6, TNF-α in atherosclerosis caused by diabetic vascular complication.Methods：1Cultured rat thoracic aortic vascular smooth muscle cell lines A7r5synchronized G0/Gl period and then divided into four groups:(1) Contorl group (NG group,5.5mmol/L glucose)(2) High Glucose group (HG group,25mmol/L glucose)(3) NG+Inhibitor LY294002groups (10,20,30μmol/L)(4) HG+Inhibitor LY294002groups (10,20,30μmol/L)2The effect of different concentrations of LY294002on the cellproliferating viability incubated in high glucose was observed by MTT assay.3The effect of LY294002on Akt, p-Akt protein expression in VSMCsincubated in high glucose was detected by Western blot.4The effect of LY294002on AP-1binding activity in VSMCs incubatedin high glucose was detected by EMSA.5The contents of IL-6and TNF-α in the supernatant were measured withthe method of ELISA.6The contents of CRP in the supernatant were measured with themethod of RIA.Results:1The effects of PI3K/Akt inhibitor LY294002proliferation viability bydifferent concentrations on VSMCs in High glucose. The result of proliferation activity VSMCs by MTT colorimetric assayshows:(1) Compared with the control group, the proliferation activity ofVSMCs induced by the high glucose was notably increased (P<0.05).(2)Compared with the normal glucose group, the proliferation activity of VSMCsinduced by the LY294002(10,20,30μmol/L) in high glucose was notablydecreased (P<0.05).(3) Inhibitor LY294002(10,20,30μmol/L) can inhibitthe proliferation of VSMCs induced by high glucose in a dose-dependentmanner (P<0.05), pairwise comparison was statistically significant betweengroups (P <0.05).2The effect of CRP lelvels by different concentrations PI3K/Aktinhibitor LY294002on VSMCsRIA detection results show:(1) The HG group CRP level weresignificantly increased, there is a statistically significant difference (P<0.05).(2) HG+different concentrations of LY294002(10,20,30μmol/L) groupscompared with the control group, no significant difference (P>0.05).(3)HG+different concentrations of LY294002(10,20μmol/L) group comparedwith HG group, CRP levels increased; LY29400210μmol/L compared withthe high-sugar group was no statistical difference (P>0.05), LY294002(20,30μmol/L), respectively, compared with the high-sugar group there is astatistically significant difference (P<0.05). LY294002（20,30μmol/L）,respectively compared with the HG group, there is a statistically significantdifference (P<0.05).(3) HG+LY29400230μmol/L of CRP level slightlyhigher than the HG+LY29400220μmol/L group, but between the two groupswas no significant difference (P>0.05).3The effect of IL-6, TNF-α lelvels by different concentrations PI3K/Aktinhibitor LY294002on VSMCELISA detection results show:(1) HG group, HG+differentconcentrations of LY294002(10,20,30μmol/L) group IL-6levels weresignificantly increased then control group (P<0.05).(2) HG+differentconcentrations of LY294002(10,20μmol/L) group IL-6levels and highglucose group, were significantly reduced, there is a statistically significant difference (P<0.05).(3)Between HG+different concentrations of LY294002(10,20,30μmol/L) groups, IL-6levels in a dose-dependent reduced withLY294002concentration increased, Pairwise comparisons showed,20μmol/Lgroup compared with10μmol/L group IL-6levels have reduced, but nosignificant difference (P>0.05), remaining pairwise comparisons werestatistically significant (P<0.05).ELISA detection results show:(1) HG group, HG+differentconcentrations of LY294002(10,20μmol/L) group TNF-α lelvel were higherthan the control group (P<0.05); HG+LY29400230μmol/L group comparewith the control group, no significant difference (P>0.05).(2) HG+differentconcentrations of LY294002(10,20,30μmol/L) of TNF-α were lower thanthe HG group, a statistically significant difference (P<0.05).(3) betweenHG+different concentrations of LY294002(10,20,30μmol/L) groups,WithLY294002concentration elevated, the TNF-α levels dose-dependently reduced,Pairwise comparisons between groups were statistically significant (P<0.05).4The effect of PI3K/Akt inhibitor LY294002of different concentrationson VSMCs for the Akt and p-Akt Ser473protein expression in high glucoseWestern blot detection results show:(1) HG, HG+LY294002(10,20,30μmol/L) expressing Akt compared with control group were not significantlydifferented (P>0.05).(2) p-Akt Ser473protein expressing in HG wassignificantly higher than that in the control group (P<0.05); HG+differentconcentrations of LY294002group (10,20,30μmol/L) p-Akt Ser473proteinexpressing was significantly increased, and the difference was statisticallysignificant (P<0.05); HG+different concentrations LY294002(10,20,30μmol/L) for pairwise comparisons between groups was statistically significant(P<0.05), with increasing inhibitor concentration, p-Akt Ser473proteinexpression in a dose-dependent Reduced.5The effect of PI3K/Akt inhibitor LY294002by different concentrationson VSMC of AP-1binding activity in high glucoseEMSA detection results show:(1) the HG group of AP-1binding activitywas significantly higher than that in the control group, the difference was statistically significant (P<0.05).(2)HG+different concentrations ofLY294002(10,20,30μmol/L) AP-1binding activity was significantly lowerthan the HG, the difference was statistically significant (P <0.05). And AP-1binding activity reduced in a dose-dependent inhibition, LY29400230μmol/Lgroup is the Significant compared with the control group (P>0.05); BetweenHG+different concentrations of LY294002(10,20,30μmol/L) grouppairwise comparison, the difference was statistically significant (P <0.05).Conclusions:1The VSMCs cultured in vitro from rat thoracic aortic can stimulated byhigh glucose proliferated and expressing of inflammatory factors as CRP, IL-6,TNF-α.2High glucose can make the p-Akt ser473phosphorylated, and activatedPI3K/Akt signaling pathway.3Nuclear transcription factor AP-1can be active by high glucose, whichwas involved in the proliferation of VSMCs and expression of inflammatoryfactors such as CRP, IL-6, TNF-α.4By inhibiting of the PI3K/Akt signal pathway the activation of AP-1was inhibited deeply.The proliferation of VSMCs decreased, and also theexpressing level of inflammatory factors such as CRP, IL-6, TNF-α.PI3K/Akt/AP-1signal pathway may play an important role in the regulation ofproliferation of VSMCs and the expression of CRP, IL-6, TNF-α.