| Objective:Macrovascular disease is one of the most common complications of diabetes mellitus(DM),and its pathophysiological basis is atherosclerosis(AS),and hyperglycemia is a high-risk factor for the formation of AS.DM belongs to the category of "xiaoke" in Traditional Chinese Medicine,whose pathogenesis is yin deficiency and dryness heat,and blood stasis exists the entire process of the disease.The key pathological basis of diabetic macrovascular disease is blood stasis;thus,promoting blood circulation and removing blood stasis are the key treatments for diabetic macrovascular disease.Studies showed that Pollen Typhae has anti-DM and anti-AS functions,and inhibits the proliferation of vascular smooth muscle cells(VSMCs).However,the potential mechanism of Pollen Typhae’s anti-diabetic atherosclerosis is kept unclear.The study aims to observe the effects of Pollen Typhae extract including Pollen Typhae total flavone(PTF),Typhaneoside(TYP)and Isorhamnetin-3-O-neohesperidoside(I3ON)on the high-glucose-induced proliferation of VSMCs,and on the cell morphology and autophagy relative protein expression level of VSMCs,which will reveal the mechanisms of Pollen Typhae inhibiting proliferation of VSMCs.Method:1.After treatment with different concentrations of glucose(0.0 m M,3.0 m M,5.0 m M,10.0 m M,20.0 m M,30.0 m M,40.0 m M)for 24 h,48 h and 72 h,respectively,VSMCs proliferation was analyzed by CCK-8 and the cell state was using under microscope.2.After treatment with different concentrations of Pollen Typhae extract(0.0625 mg/ml,0.125 mg/ml,0.25 mg/ml,0.5 mg/ml PTF;10.0 μM,20.0 μM,40.0 μM,80.0 μM,100.0 μM TYP;10.0 μM,20.0 μM,40.0 μM,80.0 μM,100.0 μM I3ON)for 24 h,48 h and 72 h,respectively,VSMCs viability was detected by CCK-8.3.After treatment with different concentrations of Pollen Typhae extract(0.0625 mg/ml,0.125 mg/ml,0.375 mg/ml PTF;10.0 μM,20.0 μM,40.0 μM,60.0 μM TYP;10.0 μM,20.0 μM,40.0 μM,60.0 μM,80.0 μM I3ON)for 24 h,48 h and 72 h in high glucose induced VSMCs,proliferation was analyzed by CCK-8.4.After treatment with TYP,rapamycin,TYP+Compound C,Compound C for 48 h in a high glucose induced VSMCs,the morphological changes of autophagy were observed by transmission electron microscope,western blot was used to detect the autophagy relative proteins(ULK1,LC3,p62)expression level of VSMCs,and VSMCs proliferation was analyzed by CCK-8.Results:1.The effect of glucose on the proliferation of VSMCsAfter treatment with different concentrations glucose for 24 h,48 h and 72 h,respectively,compared with 0.0 m M glucose group,the absorbance and cell number in the range of 3.0～40.0 m M glucose of VSMCs were significantly increased(P < 0.05 or P < 0.01).The absorbance and cell number were positively correlated to glucose concentration.The results show that glucose promotes proliferation of VSMCs in a concentration-dependent manner.2.Effect of Pollen Typhae extract on viability of vascular smooth muscle cells(1)Compared with the CON group,0.1% DMSO treatment did not change viability of VSMCs after treatment for 24 h,48 h and 72 h(all P>0.05).(2)Compared with the CON group or the 0.1% DMSO group,0.0625mg/ml,0.125 mg/ml,0.25 mg/ml,0.5 mg/ml PTF treatment did not change viability of VSMCs after treatment for 24 h,48 h and 72 h(all P>0.05).(3)Compared with the CON group or the 0.1% DMSO group,10.0μM,20.0 μM,40.0 μM TYP treatment did not change viability of VSMCs after treatment for 24 h(all P>0.05).However,80.0μM,100.0 μM TYP treatment significantly increased viability of VSMCs after treatment for 24h(both P<0.01).Compared with the CON group or the 0.1% DMSO group,10.0 μM,20.0 μM,40.0 μM,80.0 μM,100.0 μM TYP treatment did not change viability of VSMCs after treatment for 48 h and 72 h(all P>0.05).(4)Compared with the CON group or the 0.1% DMSO group,10.0 μM,20.0 μM,40.0 μM,80.0 μM,100.0 μM I3 ON treatment did not change viability of VSMCs after treatment for 24 h and 72 h(all P>0.05).Compared with the CON group,10.0 μM,20.0 μM,40.0 μM I3 ON treatment did not change viability of VSMCs after treatment for 48 h(all P>0.05),but 80.0 μM,100.0 μM I3 ON treatment slightly decreased viability of VSMCs after treatment for 48 h(both P<0.05).Compared with the 0.1% DMSO group,10.0 μM,20.0 μM,40.0 μM and 100.0 μM I3 ON treatment did not change viability of VSMCs after treatment for 48 h(all P>0.05),but 80.0 μM I3 ON treatment slightly reduced viability of VSMCs after treatment for 48 h(P<0.05).3.The effect of pollen typhae extract on VSMCs proliferation in high glucose environment(1)Compared with the 5.0 m M glucose group,30.0 m M glucose treatment significantly increased absorbance and cell number of VSMCs after treatment for 24 h,48 h and 72 h(all P<0.01),indicating the successful construction of VSMCs proliferation model.(2)Compared with the 30.0 m M glucose group,0.0625 mg/ml,0.125mg/ml,0.375 mg/ml PTF treatment significantly decreased absorbance and cell number of VSMCs in 30.0m M glucose environment after treatment for24 h,48 h(all P<0.01).In the same way,0.125 mg/ml PTF treatment for72 h also significantly reduced absorbance and cell number in highglucose-induced VSMCs(both P<0.05).(3)Compared with the 30.0m M glucose group,10.0 μM,20.0 μM,40.0 μM,60.0 μM TYP treatment decreased absorbance and cell number of VSMCs induced by 30.0 m M glucose environment after treatment for24 h,which were obviously in the 20.0 μM and 40.0 μM TYP concentration groups(P<0.05 or P< 0.01).Compared with the 30.0 m M glucose group,10.0 μM,20.0 μM,40.0 μM,60.0 μM TYP treatment remarkably decreased absorbance and cell number of VSMCs in 30.0 m M glucose environment after treatment for 48 h and 72 h in a dose-dependent fashion(all P<0.01),among which,the reduce in the absorbance and cell number in the 40.0 μM TYP group was the biggest.(4)Compared with the 30.0 m M glucose group,10.0 μM,20.0 μM,40.0 μM,60.0 μM,80.0 μM I3 ON treatment did not change absorbance or cell number of VSMCs in 30.0 m M glucose environment after treatment for 24 h,48 h and 72 h(all P>0.05).4.The effect of TYP on autophagy of VSMCs under high glucose environment(1)Compared with the MOD(30.0 m M glucose)group,a large number of autophagolysosomes exist in the cytoplasm of VSMCs in the TYP and the Rap groups;the CON group had a few autophagolysosomes.And the number of autophagolysosomes was rare and similar on TYPcc group treatment with TYP with 10.0 μM Compound C and the MOD group.There was no typical autophagy structure in CC group(treatment with Compound C).(2)Compared with the 5.0 m M glucose group,the ratio of LC3 II/LC3 I and the expression level of ULK1 protein were obviously decreased(both P<0.01)in the 30.0 m M glucose group,and the expression level of p62 protein was significantly increased(P<0.01),indicating that high glucose inhibits authagy of VSMCs.Compared with the 30.0 m M glucose group,TYP and rapamycin treatment for 48 h remarkably increased the ratio of LC3 II/LC3 I(P<0.05),the expression level of ULK1(P<0.01 or P<0.05),and significantly down-regulated the expression level of p62(P<0.01)in high glucose-induced VSMCs.Compared with the TYP group,after treatment with TYP+Compound C for 48 h,the ratio of LC3 II/LC3 I and the expression level of ULK1 were decreased significantly(P<0.05 or P<0.01),and the expression level of p62 protein was significantly increased(P<0.01)in high glucose-induced VSMCs.Implying that the promotion of TYP on autophagy of VSMCs was inhibited.(3)Compared with the 30.0 m M glucose group,40.0 μM TYP and100.0 n M rapamycin treatment for 48 h significantly decreased absorbance and cell number(all P<0.01)of VSMCs in 30.0 m M glucose environment,and the cell distribution was sparse under the microscope.Compared with the TYP group,TYP+Compound C treatment for 48 h significantly increased absorbance and cell number(both P<0.05)of VSMCs in 30.0m M glucose environment,but the absorbance and cell number were lower than that the 30.0 m M glucose concentration group(both P<0.01).Conclusion:1.Glucose promotes proliferation of VSMCs in a concentrationdependent manner.2.PTF at the concentration of 0.0625～0.375 mg/ml,inhibits highglucose-induced proliferation of VSMCs,TYP at the concentration of 10.0μM,20.0～60.0 μM inhibits the proliferation of VSMCs in a high glucose environment,10.0～80.0 μM I3 ON has no significant effect on highglucose-induced proliferation of VSMCs.3.TYP promotes autophagy of high-glucose-induced VSMCs,which is related to AMPK signaling pathway.Inhibition of TYP on high glucoseinduced proliferation of VSMCs is partially blocked by AMPK inhibitor.In short,PTF inhibits high glucose-induced proliferation of VSMCs,and TYP inhibits high glucose-induced proliferation of VSMCs partly through inducing autophagy of VSMCs.The results revealed the partial mechanisms of Pollen Typhae extract inhibiting high glucose-induced proliferation of VSMCs,which provided a scientific basis for the treatment of diabetic atherosclerosis using Pollen Typhae. |
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