Roles Of Transient Receptor Potential Canonical In Carotid Artery Remodeling Of Spontaneously Hypertensive Rat

Vascular remodeling, a sign of target organ damage caused by hypertension,is the structural basis of maintain and deterioration of hypertension,and its main pathological change is abnormal proliferation of vascularsmooth muscle cells （VSMC）. Intracellular calcium ion concentration([Ca²⁺]i) increasing continuously and slowly has been found crucial to cellproliferation. Previous studies showed that Transient Receptor PotentialCanonical （TRPC） had been found as a part of Store-operated calciumchannel （SOCC） and Receptor-operated calcium channel （ROCC）, which wasimportant to intracellular calcium steady state.Researches on hypertension vascular remodeling mainly focus on aortathoracic/abdominals and mesenteric artery, and less is known from carotidartery. It is not clear how TRPC proteins express during carotid arteryremodeling in hypertension, and whether inhibited TRPC can reducevascular remodeling.The expression of TRPC and its downstream signal nuclear factor ofactivated T cell3（NFATc3） on both the remodeling of carotid artery inspontaneously hypertensive rat （SHR） and the cultured VSMC derived fromcarotid artery of SHR will be investigated in the study. The effects andthe possible mechanisms of Ginsenoside Rb1on the remodeling of carotidartery in SHR and the cultured VSMC from carotid artery of SHR will alsobe explored. The study may provide a new therapy target at hypertensionand hypertensive vascular remodeling. Therefore, it may play an importantrole in prevention and treatment for hypertension and its arteryremodeling. Part oneThe dynamic expression of TRPC subtypes on carotid artery ofspontaneously hypertensive rat （SHR）Object: To study the expression of TRPC subtypes on carotid arteryremodeling of SHR，and explore the relationship between the expressionof TRPC and the remodeling of carotid artery of hypertension.Methods:30male SHR were randomly divided to3groups （n=10）:4weeks（4w）,8weeks （8w） and18weeks （18w） old, the age matched male WKY ascontrol group respectively（n=10）.（1） Systolic blood pressure （SBP） wasmonitored by the standard tail-cuff method biweekly.（2） Media thickness（MT）, lumen diameter （LD）, medial area （MA）, collagen area and nucleararea of carotid artery tissue sections were measured by histopathologicalstaining.（3） Carotid artery ring function was tested with the agonistand inhibitor of TRPC.（4） The expressions of subtypes of TRPC mRNA andprotein on carotid artery were measured with Real-time PCR and Westernblot respectively.Results:（1） The SBP, MT, LD, MT/LD, MA and collagen area of SHR groupincreased gradually with age.（2） Treated with noradrenalin （NE） theagonist of TRPC, the contraction of carotid artery ring in18w SHR wasstronger than other groups. On the contrary, treated with SKF96365thenonselective inhibitor of TRPC, the suppression ratio of vascularcontraction was more obvious in18w SHR than other groups.（3）Both mRNAand protein expression of TRPC1and TRPC6on carotid artery in SHR wereunregulated gradually with age.Conclusions:（1） Remodeling of the carotid artery of SHR is ingravescencewith age.（2）Unregulated TRPC1and TRPC6expressions on the carotid arteryin SHR are associated with the remodeling of carotid artery ofhypertension. Part twoProliferation and expression of TRPC1and TRPC6on VSMC derived fromcarotid artery of SHRObject: To study the expressions of TRPC1, TRPC6and downstream signalNuclear Factor of Activated T-cells3（NFATc3） as well as [Ca²⁺]i on VSMCderived from carotid artery SHR intervened with AngⅡ, and explore theroles of TRPC in VSMC proliferation of hypertension.Methods: VSMC were cultured from carotid artery of13-14week old maleSHR. VSMC with and without AngⅡ at the concentration of10-7M asexperiment group and control group respectively.（1）VSMC proliferationwas evaluated with WST-1and BrdU ELISA.（2）Both mRNA and proteinexpression of TRPC1, TRPC6and NFATc3on VSMC were tested by Real-timePCR and Western blotting respectively.（3）[Ca²⁺]i of VSMC was measuredwith con-focal microscopy.Results:（1） Both WST-1metabolism activity and BrdU-DNA synthesizeincorporation of VSMC from the experiment group were higher than thecontrol group.（2）When compared to the control group, both mRNA andprotein expressions of TRPC1, TRPC6and NFATc3on VSMC of experiment groupincreased significantly.（3）[Ca²⁺]i in VSMC of experiment group was higherthan that in the control group.Conclusion:（1） Both mRNA and protein expressions of TRPC1, TRPC6andNFATc3as well as [Ca²⁺]i increased in proliferative VSMC.（2） AngⅡ mightpromote TRPC channel upregulation, cause [Ca²⁺]i increasing, leading toVSMC proliferation. Part threeEffects of Ginsenoside Rb1on the proliferation and TRPC expression ofthe cultured VSMCObject: To study the effects of Ginsenoside Rb1on the proliferation andexpressions of TRPC1, TRPC6, NFATc3of the cultured VSMC intervened withAngⅡ，and explore the role of Ginsenoside Rb1on VSMC proliferation.Methods: VSMC with AngⅡ and Ginsenoside Rb1at concentrations of10-5M,10-6M,10-7M, and10-8M were as experiment groups, and L-voltage-dependentcalcium channel （L-VDCC） blocker-amlodipine, Ang Ⅱ receptor antagonisttelmisratan and nonselective TRPC channel inhibitor-SKF96365were ascontrol groups respectively.（1） VSMC proliferation was evaluated withWST-1and BrdU ELISA.（2） Both mRNA and protein expressions of TRPC1,TRPC6, NFATc3and L-type Ca++CPα1c （L-CPα1c） on VSMC were measured withReal-time PCR and Western blotting.（3）[Ca²⁺]i was tested with con-focalmicroscopy.Results:（1） WST-1metabolism activity and BrdU-DNA synthesize of VSMCreduced significantly,the expressions of TRPC1, TRPC6and NFATc3mRNAand protein on VSMC and [Ca²⁺]i in VSMC decreased in the experiment groupsof Ginsenoside Rb1at the concentration of10-7M, telmisartan and SKF96365group （p<0.01）.（2） The expressions of L-CPα1c mRNA and protein on VSMCreduced in Amlodipine group. However, both the expressions of TRPC1andTRPC6on VSMC and [Ca²⁺]i in VSMC were not different with or withoutAmlodipine treatment.Conclusions:（1）Ginsenoside Rb1, telmisartan attenuate VSMCproliferation，which mechanism may be that Ginsenoside Rb1, telmisartandownregulate the expression of TRPC1, TRPC6and NFATc3on VSMC and reduce[Ca²⁺]i in VSMC.（2） Amlodipine does not affect TRPC expression and cellproliferation of VSMC. Part fourEffects of Ginsenoside Rb1on carotid artery remodeling of SHRObject: To investigate the effects of Ginsenoside Rb1and the expressionsof TRPC1, TRPC6， NFATc3and L-CPα1c on carotid artery remodeling in SHR,and explore the role of Ginsenoside Rb1on carotid artery remodeling inhypertension.Methods: Forty8w SHR male rats were divided randomly into following4groups （n=10）.（1） SHR group （S）, gavaged with2ml/d distilled water,（2）Amlodipine treatment group （A）, gavaged with10mg/kg.d amlodipinebesylate,（3） Telmisartan treatment group （T）, gavaged with8mg/kg.dtelmisartan,（4） Ginsenoside Rb1treatment group （R）, intraperitonealinjection of5mg/kg.d. Age and sex matched WKY were served as control group（W, n=10）, gavaged with2ml/d distilled water. Experiment period was10weeks, and SBP was measured every2weeks. At the end of the experiment,（1） blood samples were obtained from abdominal aorta, plasma Ang Ⅱconcentration was tested with radio-immunity assay,（2） MT, LD, MA andcollagen area were measured in VEG-stained carotid artery sections, whilenuclear area was evaluated with HE staining.（3）Both mRNA and proteinexpression of TRPC1, TRPC6, L-CPα1c and NFATc3on carotid artery weretested by Real-time PCR and Western blotting.Results:（1） Blood pressure in group R decreased significantly comparedto group S, but higher than that in group A and T.（2） Plasma AngⅡ levelof group S was higher than that in group R, but lower than group T.（3）MT, MT/LD,MA, nuclear area and collagen area of Group R and T were lessthan those of group S, and no change happened to group A.（4） Group R andT had a lower expression of TRPC1and TRPC6when compared to group S; GroupR and group A had a decreasing L-CPα1c mRNA and protein expression oncarotid artery; NFATc3mRNA and protein expression decreased in threegroups. Conclusions:（1） Ginsenoside Rb1can reduce blood pressure and plasma AngⅡ concentration as well as improve carotid artery remodeling of SHR.（2） Telmisartan not only lower SBP but also reduce the degree of carotidartery remodeling of SHR.（3） Amlodipine has not effect on carotid arteryremodeling of SHR in spite of its antihypertensive effect.（4） Themechanisms that Ginsenoside Rb1and Telmisartan rather than Amlodipinecan improve carotid artery remodeling of SHR may be through downregulationof TRPC on carotid artery and reducing AngⅡof SHR. Therefore, TRPC playsan important role in carotid artery remodeling in hypertension.