| Objective: In this study,we verified that San Jie Tong Mai Fang is effective in patients with carotid atherosclerosis(CAS)through clinical observation and provided new ideas and methods for the prevention and treatment of CAS;through animal experiments,we elucidated the intrinsic mechanism of San Jie Tong Mai Fang in intervening CAS and provided experimental basis for clinical practice.Methods:1.Clinical observationIn this study,72 patients who met the inclusion criteria were collected and randomly divided into treatment group and control group.The control group was treated with atorvastatin calcium tablets,and the treatment was treated with San Jie Tong Mai Fang based on atorvastatin calcium tabletst.After 12 weeks of continuous treatment,the total scores of traditional Chinese medicine(TCM)in the two groups were assessed;the clinical efficacy of TCM was compared between the two groups;the blood lipid levels [total cholesterol(TC),triglyceride(TG),low-density lipoprotein-cholesterol(LDL-C),and high-density lipoprotein-cholesterol(HDL-C)] of the two groups were measured by automatic biochemical analyzer;the carotid artery intima-media thickness(IMT)and Crouse score of carotid plaques of the two groups were assessed by color Doppler ultrasound;the serum levels of oxidized low-density lipoprotein(ox-LDL),inflammatory factors [tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-1β] and endothelin(ET)-1,nitric oxide(NO)of the two groups were measured by enzyme-linked immunosorbent assay(ELISA).2.Experimental studyIn this experiment,we used purebred Sprague-Dawley(SD)rats as experimental subjects,half male and half female,and kept them in separate cages.After 1 week of feeding,the rats were randomly divided into seven groups: the blank group,atherosclerosis(AS)model group,simvastatin group,low-dose San Jie Tong Mai Fang group,medium-dose San Jie Tong Mai Fang group,high-dose San Jie Tong Mai Fang group,and p38 mitogen-activated protein kinase(MAPK)pathway inhibitor group(10 rats in each group).The common carotid artery was injured with a balloon in all groups except for the blank group,in which the external jugular artery was ligated but balloon injury was not induced.The rats in all groups except the sham operation group,which were still given a normal diet,were fed a high-fat diet.After 6 weeks of feeding,the low-,medium-and high-dose San Jie Tong Mai Fang groups were given the drug at doses of 16 g/kg/d,32g/kg/d and 64 g/kg/d,respectively;the simvastatin group was given 1 mg/kg/d simvastatin tablets;the pathway inhibitor group was given the p38 MAPK pathway inhibitor SB203580 at a concentration of 100 mg/kg/d by gavage.The blank group and AS model group were given an equal volume of normal saline once daily for 12 weeks.At the end of the experiment,the animals were sacrificed and samples were taken.Hematoxylin-eosin(HE)and Masson staining were used to observe the pathological changes of common carotid artery;multifunctional microplate reader was used to detect the serum levels of TC,TG,LDL-C,HDL-C;ELISA was used to detect the serum levels of ox-LDL,TNF-α,IL-6,IL-1β,ET-1,and NO;quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the gene expression levels of p38 MAPK,nuclear factor-kappa B(NF-κB),cysteinyl aspartate-specific proteinase(caspase)-3,and caspase-9 in common carotid artery tissues;and western blot was used to detect the protein expression levels of p38 MAPK,NF-κB,caspase-3,and caspase-9 in common carotid artery tissues.Results:1.Clinical observation(1)The results of TCM scores comparison between the two groups showed that the total TCM scores of the two groups before intervention were equivalent.After drug intervention,the total scores of TCM in both groups decreased,and there was significant statistical significance compared with that before intervention(P < 0.05).The total score of TCM in the treatment group was significantly lower than that in the control group(P <0.05).(2)The results of comparison of TCM clinical efficacy between the two groups showed that the overall response rate was 69.44% and the significant rate was 25.00% in the control group;the overall response rate was 82.35% and the significant rate was 41.18% in the treatment group.The effective rate and effective rate were compared between the two groups.The treatment was superior to the control group,and there was significant difference between the two groups(P < 0.05).(3)The results of the comparison of blood lipid levels between the two groups showed that lipid levels were comparable between the two groups before the intervention.After the intervention,TC,TG,and LDL-C were significantly decreased and HDL-C was significantly increased in both groups(P < 0.05).The blood lipid level after intervention was compared between the two groups.The treatment group was significantly better than the control group in reducing TC,TG and LDL-C(P < 0.05).The treatment group was increased in increasing HDL-C,but the difference was not statistically significant(P >0.05).(4)The comparison of IMT and Crouse score between the two groups showed that there was no significant difference in IMT levels and Crouse score of carotid atherosclerotic plaques between the two groups before intervention.After intervention,IMT and Crouse score were improved in different degrees in both groups(P < 0.05 or P < 0.01).The IMT and Crouse score levels after intervention were compared between the two groups,and the treatment group was significantly better than the control group(P < 0.05).(5)The results of the comparison of ox-LDL levels before and after treatment between the two groups showed that there was no significant difference in serum ox-LDL levels between the two groups of patients before the intervention.After the intervention,the serum ox-LDL levels in both groups were significantly lower than those before the intervention(P< 0.05).The serum ox-LDL level after intervention was compared between the two groups,and the treatment group was significantly better than the control group(P < 0.05).(6)The results of the comparison of TNF-α,IL-6,and IL-1β levels before and after treatment between the two groups showed that there was no significant difference in serum TNF-α,IL-6,and IL-1β levels between the two groups of patients before the intervention.After the intervention,the serum levels of TNF-α,IL-6,and IL-1β in both groups were significantly lower than those before the intervention(P < 0.05 or P < 0.01).The serum levels of TNF-α,IL-6,and IL-1β after intervention were compared between the two groups,and the treatment group was significantly better than the control group(P < 0.05).(7)The results of the comparison of ET-1/NO levels before and after treatment between the two groups showed that there was no significant difference in serum ET-1/NO levels between the two groups before intervention.After the intervention,the serum ET-1levels in both groups were significantly lower than those before the intervention(P < 0.05 or P < 0.01),and the NO levels were significantly higher than those before the intervention(P < 0.05 or P < 0.01).The serum ET-1/NO levels after intervention were compared between the two groups,and the treatment group was significantly better than the control group(P < 0.05).2.Experimental study(1)Pathological changes of common carotid artery:(1)The results of HE staining showed that compared with the blank group,the intima of the common carotid artery lesions in the model group was elevated,the structure was significantly destroyed,lipid deposition was observed,medial smooth muscle cells migrated into the subendothelial space and proliferated,foam cells and lipid components were observed in the media,and the layer of medial elastic fiberboard was decreased and arranged in a wavy pattern.Compared with the model group,the pathological changes of the common carotid artery in the simvastatin group and pathway inhibitor group were significantly alleviated,and the pathological changes of the common carotid artery in the low-,medium-,and high-dose groups of San Jie Tong Mai Fang were also improved,and the improvement was more significant in the high dose group,which was comparable to that in the inhibitor simvastatin group.(2)The results of Masson staining showed that compared with the blank group,the intima of the common carotid artery in the model group was not smooth,tortuous and undulating,the medial elastic fiberboard showed multi-layered wavy undulations,fissures were observed in the fiberboard interstitium,there was local breakage,and foam cell distribution was observed in it.Compared with the model group,the intima of the common carotid artery in the simvastatin group and pathway inhibitor group was smooth,the wavy fluctuation of the elastic fiberboard in the medial layer was significantly improved,small fissures were observed in the local medial layer,and a small amount of foam cell distribution was observed.The pathological changes of the common carotid artery in the low-,medium-,and high-dose groups of San Jie Tong Mai Fang were inhibited,and the inhibition was most significant in the high dose group,which was comparable to that in the inhibitor group and simvastatin group.(2)The results of blood lipid levels determination showed that compared with those in the blank group,the serum TC,TG,LDL-C levels in the model group were extremely significantly increased(P<0.001),and the HDL-C level was extremely significantly decreased(P<0.001).Compared with those in the model group,the serum TC,TG and LDL-C levels in the simvastatin group were extremely significantly decreased(P<0.001),and the HDL-C level was extremely significantly increased(P < 0.001).The serum TC,TG and LDL-C levels were significantly decreased in the low-,medium-and high-dose groups of San Jie Tong Mai Fang(P < 0.05 or P < 0.001),and the serum HDL-C level was significantly increased in the high-dose group(P < 0.05).(3)The results of serum ox-LDL level determination showed that compared with that in the blank group,the serum ox-LDL level in the model group was extremely significantly increased(P<0.01).Compared with that in the model group,the serum ox-LDL level in the simvastatin group was extremely significantly decreased(P<0.01).The serum ox-LDL level in the low-,medium-,and high-dose San Jie Tong Mai Fang groups were significantly decreased(P<0.05 or P<0.01),however,the decrease was more significant in the mediumand high-dose groups..(4)The results of serum inflammatory factor levels determination showed that compared with those in the blank group,the levels of serum TNF-α,IL-1β and IL-6 in the model group were significantly increased(P<0.01).Compared with those in the model group,the serum TNF-α,IL-1β and IL-6 levels in the simvastatin group and the pathway inhibitor group were extremely significantly decreased(P<0.01),the serum levels of TNF-α,IL-1β and IL-6 in the middle-dose San Jie Tong Mai Fang group decreased significantly(P<0.05 or P<0.01).The serum levels of TNF-α,IL-1β and IL-6 in the high-dose San Jie Tong Mai Fang were extremely significantly decreased(P<0.01).(5)The results of serum ET-1/NO level determination showed that compared with that in the blank group,the serum ET-1 level in the model group was extremely significantly increased(P<0.01),and the NO level was extremely significantly decreased(P<0.01).Compared with those in the model group,the serum ET-1 levels in the simvastatin and pathway inhibitor groups were extremely significantly decreased,the NO levels were extremely significantly increased(P<0.01).The serum ET-1 levels in the low-,medium-and high-dose San Jie Tong Mai Fang group were significantly decreased(P<0.05 or P<0.01);the NO levels in the medium-and high-dose San Jie Tong Mai Fang group were significantly increased(P<0.05 or P<0.01).(6)The results of determination of p38 MAPK/NF-κB m RNA and protein expression levels in the common carotid artery showed that compared with those in the blank group,the m RNA and protein expression levels of p38 MAPK/NF-κ B in the common carotid artery in the model group were extremely significantly increased(P<0.01).Compared with the model group,the m RNA expression levels of p38 MAPK/NF-κ B in the common carotid artery of the simvastatin group and the pathway inhibitor group were significantly decreased(P < 0.01),and the m RNA expression levels of p38 MAPK/NF-κB in the low-,medium-,and high-dose groups of San Jie Tong Mai Fang were decreased,with the most significant decrease in the high-dose San Jie Tong Mai Fang group(P < 0.01).Compared with the model group,the protein expression levels of p38 MAPK/NF-κB in the common carotid artery of the simvastatin group and the pathway inhibitor group were significantly decreased(P < 0.01),and the protein expression levels of p38 MAPK/NF-κB in the low-,medium-,and high-dose groups of San Jie Tong Mai Fang were decreased,with the most significant decrease in the high-dose San Jie Tong Mai Fang group(P < 0.01).(7)The results of determination of caspase-9/caspase-3 m RNA and protein expression levels in the common carotid artery showed that compared with those in the blank group,the m RNA and protein expression levels of caspase-9/caspase-3 in the common carotid artery tissue in the model group were extremely significantly increased(P < 0.01).Compared with the model group,the m RNA expression levels of caspase-9/caspase-3 in the common carotid artery were significantly decreased in the simvastatin group and the pathway inhibitor group(P < 0.01),significantly decreased in the low-and medium-dose groups of San Jie Tong Mai Fang(P < 0.05),and significantly decreased in the high-dose San Jie Tong Mai Fang group(P < 0.01).Compared with the model group,the protein expression levels of caspase-9/caspase-3 in the common carotid artery of the simvastatin group and the pathway inhibitor group were significantly decreased(P < 0.01),and the protein expression levels of caspase-9/caspase-3 in the low-,medium-,and high-dose groups of San Jie Tong Mai Fang were decreased,with the more significant decrease in the medium-,and high-dose San Jie Tong Mai Fang groups(P < 0.01).Conclusion:(1)In this study,clinical observations showed that San Jie Tong Mai Fang was effective in patients with carotid atherosclerosis and could provide new ideas and methods for the prevention and treatment of atherosclerosis.(2)In this study,animal experiments showed that the mechanism of San Jie Tong Mai Fang in intervening carotid atherosclerosis was at least partially attributed to adjusting blood lipid levels and inhibiting p38 MAPK/NF-κB signaling pathway,so as to achieve anti-inflammatory,anti-apoptotic,and protect vascular endothelial function. |
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