| Ojective:To evaluate the clinical efficacy of Shugan Wendan decoction in the treatment of carotid atherosclerosis;To investigate the mechanism of Shugan Wendan decoction in treating atherosclerosis based on LXR?/NF?B signal,and to provide scientific basis for the prevention and treatment of atherosclerosis by traditional Chinese medicine.Methods:1.Clinical trial: randomized,controlled trial was performed.80 carotid atherosclerosis patients who met the diagnosis of traditional Chinese medicine were randomly assigned to the experimental group and the control group in the proportion with 1:1.Both groups were given basic treatment: chronic disease management and conventional Western medicine treatment.Control group was given atorvastatin(10mg,1time/night),experimental group was given atorvastatin and Shugan Wendan dection(1 time/day,5 days/week).The parameters which we collected before and after treatment are as followed:carotid IMT,the indicators of vascular endothelial function,blood lipids,hs-CRP,fibrinogen,MMP-9 and TCM syndrome scores.The treatment period is 12 weeks.2.Animal experiments: a New Zealand rabbit model of atherosclerosis with liver-qi stagnation was established by using calf serum albumin immune injury,high fat feeding and bondage emotional stress method.Theses rabbits are randomly divided into 6 groups,6 in each group,which were model group,atorvastatin group,Shugan Wendan decoction low,medium and high dose group,control group.After successful modeling,the rabbits were treated by injecting drugs to gastric.Atorvastatin(5 mg/kg/d),low,middle and high dose Shugan Wendan decoction(2.18 g/kg/d,6.54 g/kg/d,19.62 g/kg/d)were administered respectively.The gavage volume is 4ml/kg/d.The control group and the model group were given intragastric administration of saline in the same volume.The period of gavage is 6 weeks.(1)When the administration is finished,the carotid IMT of each rabbit was examined by ultrasound.The inner diameter and blood flow velocity were measured.(2)Serum levels of TC,TG,LDL-C,HDL-C,NO,and ET-1 of the rabbits were detected by enzyme method,nitrate reductase method,and ELISA,respectively.(3)The pathological changes of the rabbits were detected by HE staining.(4)The gene expression of CRP,IL-1?,IL-6 and MMP-9 in the aorta was detected byq PCR method.(5)The protein expression of LXR?/NF?B signaling pathway wasdetected by Western blotting.3.In vitro experiments:The vascular endothelial cell injury model was established by using ox-LDL induced HUVECs.the Shugan Wendan decoction containing serum was used as intervention measure.The experimental groups were as followed: normal control group,ox-LDL group,and ox-LDL+20% Shugan Wendan Decoction serumgroup,ox-LDL+40% Shugan Wendan Decoction serumgroup,ox-LDL+60% Shugan Wendan Decoction serum group.(1)After the culture of 24 hours,the changes of foaming of HUVECs were observed by oil red O staining.(2)The contents of NO and ET-1 in supernatantswere detected by Nitrate reductase and ELISA respectively.(3)The gene expressionsof CRP,IL-1? and IL-6 in cellswere detected by q PCR.(4)The protein expression of LXR?/NF?B signal pathway was detected by Western blotting.LXR? agonists and inhibitors were used as interventions.And the experimental groups were as followed:ox-LDL group,ox-LDL+60% Shugan Wendan decoction containing serum,ox-LDL+60% Shugan Wendan Decoction serum plus LXR? Inhibitor(SR9243)group,ox-LDL+LXR? agonist(GW3965)group.(1)The level of NO and ET-1 in the culture supernatant was measured by Nitric acid reductase and ELISA.(2)The expression of CRP,IL-1?,IL-6,MMP-9 and the protein expression of LXR?/NF?B signaling pathway were detected by QPCR and Western blotting,respectively.Results:1.Clinical trial:(1)There was no statistically significant difference between the experimental group and the control group in improving carotid IMT of patients with carotid atherosclerosis(P>0.05).Compared with the control group,the levels of TC,LDL-C,hs-CRP,fibrinogen,MMP-9,and ET-1 in the experimental group decreased,and the level of NO increased,and the difference was statistically significant(P<0.05).(2)The TCM syndrome scores of the experimental group,such as thoracic hypotension,vertigo,and headache,were significantly lower than those in the control group.The markedly effective rate and total effective rate were significantly higher than those in the control group,and the differences were statistically significant(P<0.05).2.Animal experiments:(1)Ultrasound results: compared with the model group,the internal diameters of the atorvastatin group and the medium ?high dose Shugan Wendan decoction groups were widened,and the carotid IMT was decreased,the difference was statistically significant(P < 0.05).There was no statistically significant difference in blood flow rate among experimental groups(P > 0.05).(2)Pathological results: In the model group,the lumen of the blood vessels was significantly narrowed,atheromatous plaques were formed,and a large number of intracellular foam-like changes were seen.In the atorvastatin group and Shugan Wendan decoction group,the blood vessels in the high,middle,and low concentration groups were narrowed.Atherosclerotic plaques and foam-like changes were all lower than the model group.(3)Blood lipids: Compared with the normal control group,the TG,TC,and LDL-C levels in the model groupincreased,HDL decreased,and the differences were statistically significant(P<0.05).Compared with the model group,the levels of TG,TC and LDL-C the atorvastatin group andlow,medium,and highdose Shugan Wen Dan decoction group decreased,and the differences were statistically significant(P < 0.05).(4)Endothelial cell function: Compared with the normal control group,NO in the model groupdecreased,ET-1 increased,the difference was statistically significant(P < 0.05).Compared with the model group,NO in the atorvastatin group and the highdose Shugan Wen Dan decoction group increased and ET-1 decreased,the difference was statistically significant(P < 0.05).(5)QPCR results: Compared with the normal control group,the expression levels of hs-CRP,IL-1?,IL-6 and MMP-9 in the model group all increased,and the differences were statistically significant(P < 0.05).In group comparison,the expression levels of CRP,IL-1?,IL-6 and MMP-9 in the atorvastatin group,the low,middle and high dose Shugan Wendan decoction groups all decreased,and the differences were statistically significant(P < 0.05).(6)Western blotting showed that compared with the normal control group,the expression of LXR? protein in the model group was decreased,and the protein expression of NF?B was increased.Compared with the model control group,the concentrations of atorvastatin group and Shugan Wendan decoction group were higher than those of the control group.The expression of LXR? protein in the group was increased and the protein expression of NF?B was decreased.3.In vitro experiments: the results of different concentrations of Shugan Wendan Decoction containing serum intervention are as follows.(1)Oil Red O staining results showed that red lipid droplets in HUVECs of Shugan Wendan decoction containing serum decreased.And the red lipid droplets decreased with the increase of Shugan Wendan decoction concentration.(2)Compared with the normal group,the NO content in the model group decreased,and the ET-1 content increased,the differenceswere statistically significant(P < 0.05).Compared with the model group,the NO value in theox-LDL+60% Shugan Wendan Decoction containing serum group increased,the difference was statistically significant(P < 0.05).Compared with the model group,the ET-1 in the serum of the different volumes of Shugan Wendan Decoction group decreased,the differences were statistically significant(P < 0.05).(3)QPCR showed that compared with the normal control group,the expression levels of CRP,IL-1?,IL-6 and MMP-9 in the model group were increased,the differenceswere statistically significant(P<0.05).Compared with the model group,the expression of CRP,IL-1?,IL-6 and MMP-9 in the serum of Hegan Wendan decoction group was reduced,and the difference was statistically significant(P < 0.05).(4)Western blotting results showed that compared with the normal control group,the expression of LXR? protein in the model group cells was decreased,while the expression of NF?B protein was increased.Compared with the model group,the expression of LXR? proteinin Shugan Wendan Decoction serum-containing cells was increased,while the expression of NF?B protein was decreased.The results of LXR? agonist and inhibitor interventions are as follows.(1)Western blotting showed that compared with ox-LDL group,LXR? protein expressionin 60% Shugan Wendan decoction + ox-LDL group and oxLDL + LXR? agonist group was up-regulated,while the protein expression of NF?B was down-regulated.Compared with 60% Shugan Wendan Decoction + ox-LDL group,60% Shugan Wendan decoction + ox-LDL + LXR? inhibitor group showed down-regulation of LXR? protein expression and upregulation of NF?B protein expression.(2)Oil red O staining: Compared with the ox-LDL group,the number of red lipid droplets in the 60% Shugan Wendan decoction+ox-LDL group and the ox-LDL+LXR? agonist group was significantly reduced.The number of red lipid droplets in the 60% Shugan Wendan Decoction + ox-LDL+LXR? inhibitor group increased compared with the 60% Shugan Wendan Decoction + ox-LDL group.(3)Detection of NO and ET-1 in cell supernatant: Compared with ox-LDL group,NO level in oxLDL+LXR? agonist group was increased and ET-1 was decreased,the differences were statistically significant(P < 0.05).Compared with oxLDL+60% Shugan Wendan Decoction containing serum,ox-LDL+60% Shugan Wendan Decoction serum + LXR? inhibitor group decreased,NO levels and ET-1 levels increased.Differences were statistical significance(P <0.05).(4)QPCR showed that compared with ox-LDL group,the expression of CRP,IL-1?,IL-6 and MMP-9 in 60% Shugan Wendan decoction + ox-LDL group and ox-LDL + LXR? agonist group were down-regulated,and the differences were statistically significant(P<0.05).Compared with 60% Shugan Wendan Decoction+ox-LDL group,the expression of CRP,IL-1?,IL-6 and MMP-9 in the ox-LDL+60% Shugan Wendan Decoction serum+LXR? inhibitor group decreased,and the differences were statistically significant(P<0.05).Conclusions:(1)Clinical trial: Shugan Wendan Decoction can improve blood lipids,inflammation,vascular endothelial function in patients with carotid atherosclerosis,also it can increase plaque stability and inhance TCM syndromes.(2)In vivo and in vitro experiments : Shugan Wendan Decoction can inhance the function of vascular endothelial cells and the stability of atherosclerotic plaque by regulating LXR?/NF?B signaling pathway. |
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