Effects Of Compatibility Of Effective Components Of Salvia Miltiorrhiza On The Adventitia Fibroblast Cell And Spontaneously Hypertensive Rats And Its Mechanism

Part one Application of Uniform Design and Orthogonal Design in Screening the Best Compatibility of Effective Components of Salvia Miltiorrhiza in Inhibiting Fibroblast Cell ProliferationObjective: Salvia is a traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases.It contains four kinds of main water-soluble ingredients: danshensu(DSS),salvianolic acid A(Sal-A),salvianolic acid B(Sal-B)and protocatechuic aldehyde(PAL).We referred to as(SABP).A large number of studies have confirmed that each component can play a role in cardiovascular protection through different mechanisms of action.The adventitial fibroblast(AF)acts as a biological treatment center to regulate the structure and function of the blood vessels from the outside-in.The vessels are in response to stress and injury.The adventitial fibroblasts are first rapidly activated by reprogramming itself to show the behavior of different functions and structures,and is considered the starting factor for the vascular response.At present,the compatibility of traditional Chinese medicine has become a new model of traditional Chinese medicine innovation research.Therefore,we envisage the optimal combination of water-soluble components in Salvia miltiorrhiza 4 by observing its effect on the adventitial fibroblasts by uniform design and orthogonal design.Methods: In this study,adult Sprague Dawley(SD)rats were used to culture the adventitial fibroblasts of the thoracic aorta.MTT assay was used to observe the inhibitory effect of the drug on the platelet proliferation.The results were read using the microplate reader and the inhibition rate was calculated.Screening steps are as follows: 1、The initial screening Salvia powder(salvia miltiorrhiza lyophilized powder,SMLP)and four active ingredients of the application dose range.2、The use of uniform design and orthogonal design method,the composition of the dose and compatibility screening.3、The experimental combination of the optimal combination of re-arranged to verify.Results:1 Drug concentration preliminary screening results: The effective concentration range of Sal-A,Sal-B and DSS was(10-3 mol/L,10-4 mol/L,10-5 mol/L),and the effective concentration range of PAL was(10-2 mol/L,10-3 mol/L,10-4 mol/L),but SMLP had no obvious effect on adventitial fibroblasts inhibition,and there was no statistically significant difference between the concentrations;2 Uniform design of the experimental results: combined with the results of the initial screening drug concentration experiments,the four components selected effective dose range of 6 different concentrations,and then in accordance with the uniform design table that is 4 factors 6 level uniform table U6(64)to arrange the experiment,the results Showing that group 3 was the most effective combination,the most significant inhibition was statistically significant.S3(10-4 mol/L),A6(10-6 mol/L),B2(5×10-4 mol/L)and P4(5×10-4 mol/L);3 Orthogonal design experiment results: combined with the uniform design of the experimental results,in the optimal treatment,according to the corresponding dose of each component within the range of similar selection of low,medium and high three different doses of DSS,Sal-A,Sal-B,PAL four components of the effective dose of low,medium and high orthogonal design experimental study,select L9(34)orthogonal table(Table 2),the results show that the sixth group is the most significant combination of inhibition,with statistics Differences in learning.S2(1.5×10-4 mol/L),A3(7×10-6 mol/L),B1(3×10-4 mol/L)and P2(5×10-4 mol/L);4 The four ingredients of Danshen were re-arranged in combination to further verify the best compatibility ratio: According to the results of uniform design and orthogonal design,for each component to determine a most appropriate effective dose,the four components arranged again,The results showed that the inhibitory rate of SABP group was the highest,which was the best combination ratio.Conclusions: In this experiment,the four main water-soluble components(DSS,Sal-A,Sal-B,PAL)of Salvia miltiorrhiza injection were selected to optimize the value of adventitial fibroblasts by using uniform design and orthogonal design experiment Compatibility ratio(150︰7︰300︰500)and concentration: S(1.5×10-4 mol/L),A(7×10-6 mol/L),B(3×10-4 mol/L),P(5×10-4 mol/L).There are still some synergies between the four components.Part two Protective Effect of the Best Compatibility of Effective Components of Salvia Miltiorrhiza on Vascular Remodeling in Spontaneously Hypertensive RatsObjective: Vascular remodeling(VR)is the common pathologic basis of target organ damage such as heart,brain and kidney.It is manifested as vascular smooth muscle cell(VSMC)hypertrophy,hyperplasia and extracellular matrix(ECM)increased,the outer membrane thickening irregular,resulting in wall thickening hardened,decreased compliance,etc.then lead to abnormal vascular function.Angiotensin II(Ang II)and endothelin(ET)are the major vasoconstrictor factors.α-SMA is a marker of VSMC and AF transformation into myofibroblasts,Collagen Type Ⅰ(Col-Ⅰ)is the main component of ECM,they are signs of vascular structural changes.Therefore,in this study,spontaneously hypertensive rats(SHR)were used to observe the effect of SABP on blood pressure and the effect of vascular remodeling.Methods: The experimental animals were randomly divided into 5 groups:(1)normal control group,(WKY);(2)model group,(SHR);(3)perindopril(PD)group,(SHR);(4)salvia miltiorrhiza lyophilized powder(SMLP)group,(SHR);(5)SABP group(SHR).SMLP(1 g/kg/d);PD(0.4 mg/kg/d);SABP(DSS: 5 mg/kg/d,Sal-A: 0.233 mg/kg/d,Sal-B: 10 mg/kg/d,PAL: 17 mg/kg/day);SHR and WKY control group were given the same amount of saline.The tail arterial blood pressure was measured every 2 W before and during administration.After 8W administration,the serum and thoracic aorta of each animal were collected for ELISA,HE staining,immunohistochemistry,R-T PCR and Western Blot.Results:1 SABP can significantly reduce SHR systolic blood pressure,but not to the normal level.SABP group and perindopril group(PD)in the whole administration process to make blood pressure steadily decreased.There was no significant difference between the two groups in the antihypertensive effect.SMLP had no significant antihypertensive effect,and there was no significant difference compared with SHR group;2 SABP significantly reduced the levels of AngII and ET-1 in serum of SHR rats.The descending effect of AngII and ET-1 in PD group was similar to that in SABP group.There was no significant difference in the levels of Ang II and ET-1 in SMLP serum and normal SHR rats;3 SABP can significantly improve SHR thoracic aortic remodeling.HE staining showed that the thoracic aortic endothelial cells in the SHR group had the same effect on VSMC hypertrophy,the outer membrane and the wall were thickened.The thoracic aortic endothelial cells in the SABP group had no shedding,no blood cell adhesion.There was no obvious thickening of the wall.The histopathological changes of the PD group were similar to those of the SABP group and the WKY group.SMLP group is similar to SHR group;The thickness of aortic wall thickness(MT),vessel wall thickness/ radius(VT/VA),adventitia thickness of the thoracic aorta(AT)was significantly reduced by SABP than those in the SHR group.And this effect was similar to that of PD groups.The effect of SMLP on arterial structure was not significantly changed compared with SHR group;4 Immunohistochemical observation results: The expression of Col-I and ET-1 in SABP group was significantly lower than that in SHR group.The expression of Col-I and ET-1 in SABP group was similar to WKY group.However,there was no significant difference in Col-I and ET-1 between SMLP group and SHR group;5 SABP significantly reduced the expression of Col-I,ET-1 and α-SMA mRNA in rat thoracic aorta.There was no significant difference between SABP、perindopril and WKY group;6 SABP significantly reduced the expression of Col-I,ET-1 and α-SMA proteins in the thoracic aorta of SHR rats.Conclusions: SABP significantly reduced blood pressure in SHR rats,reduced serum levels of Ang II and ET-1,significantly improved the vascular remodeling of the thoracic aorta,reduced the expression of α-SMA,Col-Ⅰ and ET-1 in the SHR thoracic aorta.And reduce the thickness of the vascular wall and outer membrane.It has obvious role of antihypertensive and vascular remodeling protection.Part three Mechanism of Reducing Vascular Remodeling in Spont-aneously Hypertensive Rats by the Best Compatibility of Effective Components of Salvia MiltiorrhizaObjective: Oxidative stress is one of the important factors leading to high blood pressure,is due to excessive accumulation of reactive oxygen species(ROS)and trigger cell damage,leading to vascular remodeling.NADPH oxidase(NOX)is the main source of ROS in the cardiovascular system and is mainly concentrated on the adventitia of the blood vessels.Studies have shown that NOX4 is expressed in the entire vessel wall.Superoxide dismutase(SOD)is the main antioxidant enzyme in the body,mainly used to reflect the body’s antioxidant capacity;malondialdehyde(MDA)is usually used to express the body’s total lipid peroxidation level.Transforming growth factor-beta(TGF-β)is one of the most important factors in the regulation of AF/MF and Col-I.TGF-β/Smad makes its classical signal transduction pathway,Smad7 is an important negative regulator in this pathway.In this study,we selected SHR rats and SD rat vascular adventitial fibroblasts,to explore the impact of SABP on its oxidative stress,and observe the NOX4,TGF-β1 and Smad7 expression on the thoracic aorta in SHR.Methods: The animals were randomly divided into 5 groups:(1)normal control group(WKY);(2)model group(SHR);(3)PD group(SHR);(4)SMLP group(SHR);(5)SABP group(SHR).SABP(1 g/kg/d);PD(0.4 mg/ kg/d);SABP(DSS: 5 mg/kg/d,Sal-A: 0.233 mg/kg/ B: 10 mg/kg/day,PAL: 17 mg/kg/day);SHR and WKY control group were given the same amount of saline.After 8W administration,the serum and thoracic aorta of each animal were collected for ELISA,immunohistochemistry,R-T PCR and Western Blot.The AF of rats were divided into four groups:(1)Ang-Ⅱ group(10-6 mol/L);(2)Fluorescent control group,fluorescent agent DCFH-DA(10-5 mol/L);(3)Ang-Ⅱ+SABP group,Ang-Ⅱ drug concentration was the same as above,SABP:(DSS: 1.5×10-4 mol/L,Sal-A: 7×10-6 mol/L,Sal-B: 3×10-4 mol/L,PAL: 5×10-4 mol/L).(4)SABP group(drug concentration above).Fluorescent agents were added to each group.Fluorescence values were measured at 502 nm using a fluoroscopy and photographed with a fluorescence microscope.Results:1 SABP significantly reduced the levels of MDA and TGF-β1 in serum of SHR rats,and significantly increased the content of SOD.2 Immunohistochemical staining showed that SABP could significantly decrease the expression of NOX4 and TGF-β1 in thoracic aorta of SHR rats,and there was no significant difference in NOX4 and TGF-β1 expression compared with WKY group,the difference was not statistically significant.This effect is similar to that of the PD group.3 SABP significantly reduced NOX4 mRNA and protein expression in thoracic aorta of SHR rats.The expression of NOX4 was similar to that of perindopril and WKY.4 SABP significantly reduced the content of ROS in AF rats stimulated by Ang-Ⅱ.SABP group and Ang-Ⅱ+SABP group compared with the blank control group were no significant difference,no statistically significant.5 SABP significantly reduced the expression of TGF-β1 mRNA and protein in the thoracic aorta of SHR rats,while the expression of Smad7 mRNA and protein was significantly increased.Conclusions: SABP can significantly reduce the level of oxidative stress in SHR rats: decrease the level of MDA and increase level of SOD in SHR serum;and decrease the expression of NOX4 in thoracic aorta,and decrease the content of ROS in AF of SD rats stimulated by Ang-Ⅱ.SABP can also decrease the level of TGF-β1 expression,increased Smad7 protein expression.SABP protects the vascular remodeling of hypertensive rats by inhibiting the expression of oxidative stress and TGF-β and increased Smad7 protein expression.