| Diabetes Mellitus(DM)is a chronic disease,diabetic angiopathy(DA),the most important complicaton of DM,underlied the pathogenesis of diabetic patients' organical complications.Different grades blood vessels suffered from DA show differently, artherosclerosis in large blood vessels on earlier stage,disfunction of vasodilatation and vasoconstriction,proliferation of vascular smooth muscle cells(VSMCs),microcirculation dysfunction,microangioma,thickening basal lamina.The relationship between DA development and hyperglycaemia isn't linearity.Now,studying the mechanism of DA and developing new drugs to block or reduce pathological agents that aggravated DA have being a hot spot,based on a good control of blood glucose.There are four main mechanisms of hyperglycaemia impairing vessels as cell signal transduction research had showed abroad:①Polyhydric alcohol alternative pathway activation:esclating metabolite--D-glucitol,which accumulating in cell and leading to cellular oedema.②Calcium-phospholipid-dependent protein kinase(PKC)activation:leading to reconstruction and function changes of vessels associated closely whith DA and other diabetic chronic diseases.③Nonenzymatic conversion of protein activation:can produce advanced glycation end products(AGEs),known to accumulate during the course of diabetes, demonstrating to decrease collagen degradation,increase basilaris substantia,thicken basal lamina,low the elasticity of blood vessels walls,raise the resistance of vessels,stenosis lumens.④Oxidative stress level raised:raising the sensitivity to oxdative damage which underliering the diabetic chronic blood vessels complications.Westen medcine drugs treated the DA are PKC inhibitors,AGEs blockers,aldose reductase activity inhibitors, antioxidant.Most of these drugs are on the stage of laboratary experiment,and the safty and efficiency of the drugs are unclear.Chinese physicians consider the combination of phlegm and stagnant blood is the major mechanism of DA.The Chinese medicin differentiation of symptoms and signs of DA are:deficiency of qi and yin,dual vacuity of yin and yang.As phlegm and stagnant blood always stay in body when suffered DA,so activating blood circulation to dissipate blood stasis,dissipate phlegm and eliminating stagnation are base treatments. Tetramethylpyrazine(TMP),a simple substance extracted,from Chinese herb chuanxiong, is used to treat variety diseases with stagnant blood symptoms,including DA,in Chinese physician's clinical therapy.VSMCs,the main construction of blood vessels,proliferating leads to DA main pathologic changes:blood vessels reconstructe,stenosis lumens,disfunction of vasodilation and vasoconstriction.Large-conductance Cacilum-activated potassium channels(BKCa), abound in VSMCs,play an important role in modulating vascular smooth muscle tone. Previews reports showed that TMP could dilate blood vessels by increasing the open probability(Po),shortenning close time(Tc)of BKCa.Otherwise,TMP could inhibite vitro cultured VSMCs proliferating,attenuate the expression of apoptosis gene c-myc,make VSMCs stayed in Glstage,decrease the amount of cells in G2+M stages.The relationship between TMP,VSMCs apoptosis,BKCais rare reported abroad or internal,the experiment was disiged to identifiy their relationship.Object:To study TMP's effect in vivo on BKCa,gene expression of c-myc,Bcl-2,Bax on arotic VSMCs of early stage diabetes rats;TMP affects proliferation and apoptosis on in vitro cultured VSMCS,and pospect the possible cell signal transduction passway of TMP induced VSMCs apoptosis.Method:One large dose Streptozocine intraperitoneally injected to establish diabetic rats model.All rats were divided into four groups randomly,they were normal group,DM model group,TMP low dose group,TMP high dose group.Rats in nornal group and DM model group were intraperitioneally injected with water for injection;in TMP low dose and high dose groups,were intraperitoneally injected with TMP injection 40mg/Kg,80mg/Kg respectly,all once a day.8 weeks later,half rats in each group were killed by descapitation, aorta thoracica isolated were fixed in formalin for situ hybridization of c-myc,Bcl-2,Bax. The rest rats were killed one after another,arota thoracica ioslated were digested in enzyme solution to get single VSMC for cell attached patch-clamp technique test,a method used to record BKCacurrent amplitude,open posssibality(NPo),time of open(To),time of close (Tc).VSMCs of arota thoracica cultured in vitro were divided into different groups,added different dose TMP and/or BKCaactivated agent NS1619 incubated for 24h.Then Mono-nuclear cell direc cytotoxicity assay(MTT)were use to test their affection on VSMCs.Other VSMCs were grouped,added different agent,NS1619,GSNO,p38 inhibitor, PKC inhibitor peptide,paxilline,TMP,incubated for 24h.Hoechst3342(fluorescence)was used to stain cells which can idetifiy apoptosis cells.Counting apoptosis VSMCs and normal cells,calculating apoptosis rate.Get the results,presuming the possible cell signal transduction involved BKCa,TMP.Results:1 Reslts of STZ intraperitoneally injected to establish diabetic rats modelAmong 60 SD rats intraperitoneally injected with STZ 55mg/Kg once,46 were succeed in establish diabetic model,1 dead,achievement ratio was 76.67%,mortality ratio was 2.17%.2 Affection of TMP on BKCaof VSMCsThe NPo of BKCaincreased in all there groups compared with normal group(P=0.000), the average close time(Tc)were shorter than that in normal group(P=0.000).The NPo in TMP low and high dose groups were higher than that of DM model group(P=0.000),and the Tc was shorter than DM model group.The average open time(To)among four groups were similar(P＞0.05).The NPo of TMP high dose group was higher than that in TMP low dose group,but the Tc was shorter,there was significance between the two groups.3 Influence on the expression of apotosis related gene:c-myc,Bcl-2,BaxOptical density(OD)was use to test the expression of c-myc,Bcl-2,Bax in situ hybridization.Compared between each group,the OD was significant(P＜0.01);The expressions of c-myc,Bcl-2,Bax in normal group were lower than other three groups; the expressions of c-myc and Bcl-2 in two TMP groups is lower than those of DM model group,the Bax expressed lower than that of model group(P＜0.01).Moreover there was significance between two dose TMP groups(P＜0.01).4 Influence on in vitro cultured VSMCs growth of TMP and NS1619Calculated the VSMCs survival rate based on MTT OD,taken the survival rate of compared group as 1,compared survival rates between different groups.The survival rate of model group was exceeded 1,indicated the cell proliferation,compared with rates in other 6 groups that were low than 1 which showed the growth of cells was inhibited,there was significance(P＜0.01).Indicated that TMP and NS1619 could inhibitor the proliferation of VSMCs induced by PDGF-BB.The influence on cell proliferation in NS1619 low dose,TMP low,high dose groups were similar(P＞0.05).,in NS1619 high dose,mixed low dose,mix high dosegroups were similar(P＞0.05),in TMP high dose,low dose groups were similar too(P＞0.05).Survival rates in NS1619 high dose,mixed low dose, mixed high dose groups were 37.34±6.34%,48.93±7.03%,31.13±5.04%,were low than other groups significantly(P＜0.01),that indicated a strong poisonous effect on cells. 5 Results of study on cell signal transduction mechnism of BKCamediated VSMCs apoptosisApoptosis rate were similar between low glucose(compared group)and high glucoses groups(model groups),(10.22±0.81%vs 12.89±2.46%,P＞0.05).The rate in NS1619 low, high dose,GSNO,GSNO+ paxilline groups were higher than that of model group(P＜0.01), which show NS1619 and GSNO could induce VSMCs apoptosis,among them the effect of GSNO was powerful,and that of TMP was mild,there were significant.The BKCa blocker paxilline could decrease the apoptosis rate significantly(P＜0.05)in GSNO group, but still higher than those of NS1619 groups,TMP group,and model group(P＜0.01), which indicated that BKCainvolved the signal transduction of apoptosis induced by GSNO, moverove there were other pathways.The apoptosis rate elevated as the dose of NS1619 increasing(P＜0.01).The apoptosis rates in groups involved p38 blocker SB202190 and PKC inhibitor peptide(SB202190,SB202190+paxilline,PKC inhibitor peptide,PKC inhibitor peptide+paxilline)were similar to that of model group(P＜0.05),indicated p38,PKC had no effect on VSMCs apoptosis,and paxilline did not effect on p38,PKC signal transduction in the trial.Conclusion:The study observed that the NPo of BKCaon VSMCs was increased as compensation,TMP could enhance the NPo,in the early phase of Diabetes rats,the effect were reinforced as the dose increasing.TMP also could low the gene expression of c-myc, Bcl-2 that could inhibite cells apoptosis,and promote the expression of Bax which could accelerate cells apoptosis by dose improvement.The proliferation induced by PDGF-BB of VSMCs cultured in vitro were inhibited by TMP,and TMP could induce cells apoptosis mediated by BKCa.TMP were safety,less cytotoxicity contrasted to NS1619.The possible passway that TMP induced VSMCs apoptosis mediated by BKCaand not by BKCawere presumed as following depiction.1 Passway of cell signal transduction of VSMCs induced by TMP mediated by BKCa1.1 TMP activates BKCadirectly,that can promote IK(Ca)and intracellular potassium efflux.The decreasing of[K+]i can low down the volume of VSMCs,or raise caspase nuclease,both switch cell apoptosis respect.1.2 TMP can promote synthesis of eNOS,increase NO,which can activate BKCa directly or indirectly,then lead to VSMCs apoptosis.2 Passway of cell signal transduction of VSMCs induced by TMP not mediated by BKCa: 2.1 TMP can promote synthesis of eNOS,increase NO.NO makes mitochondrion depolarized,which induce the release of Cytochrome-C(cyt-c).Cyt-c could combined with Apaf-1,activates caspase,induces cells apoptosis.On this passway,if Bcl-2 combines with. Apaf-1,the Apaf-1 can't combined with cyt-c,and can't activate caspase.If Bax combines with Bcl-2 that would block Bcl-2 combines with Apaf-1,it will not affect Apaf-1 activates caspase and induce apoptosis.2.2 TMP promote synthesis of eNOS,increase NO.NO can induce Fas,Fas activates sphingolipase,decompose sphingolipid to acyl sphingosine which can activate caspase result in intein degeneration,cells apoptosis.
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