| Atherosclerosis(AS)is the main cause of cardiovascular disease.It has been shown that AS is the result of the imbalance of lipid metabolism and the continuous inflammatory response.It is a chronic inflammatory disease in which a large number of lipids and cholesterol accumulate and produce fibroplaques.Therefore,it is of great significance to study and elucidate the pathogenesis of AS and further discover the drugs for the treatment of AS for the prevention of AS and cardiovascular disease.Buyang Huanwu Decoction(BYHWD)is a representative prescription for Invigorating Qi,promoting blood circulation and removing blood stasis.Modern medical research has proved that BYHWD has a definite effect on cardiovascular and cerebrovascular diseases caused by AS,but its specific mechanism has not been fully elucidated.Regulatory T cells(Treg)play an important role in regulating the balance of the immune system and maintaining the stability of the internal environment.Previous studies have shown that Treg cells protect as plaques mainly by controlling immune,inflammatory response and reducing lipid deposition.However,the mechanism of BYHWD in the treatment of AS has not been reported.Therefore,in order to explain the material basis of BYHWD’s anti-AS effect,this paper takes the theory of immune inflammation as the theoretical guidance and Treg as the starting point,and puts forward the hypothesis:BYHWD can regulate TGF-β/Smad signal pathway,promote peripheral differentiation of Treg,increase its number,restore the immune balance between CD4~+T cells,regulation of lipid metabolism,inhibit inflammatory response and stabilize AS plaque.On the one hand,it provides theoretical and experimental basis for further clarifying the pharmacological effect of BYHWD;on the other hand,it discusses the protective effect and mechanism of traditional Chinese medicine on AS plaque from the perspective of immunity and inflammation,providing new ideas for clinical multi-target to stabilize plaque.On the basis of the above experimental research,chitosan solution-based vortex-forced matrix solid phase dispersion method was established and applied to the green quality evaluation of Ligustri Lucidi Fructus,which laid the foundation for the clinical application and the development of health products of Ligustri Lucidi Fructus.1.According to the methods described in the classical literature,Apo E-/-mice were fed on high-fat diet for 12 weeks to successfully replicate AS model.BYHWD was administrated by gavage for 4 weeks.Oil red O and HE staining were used to observe the effect of BYHWD on the formation of plaques in the whole aorta and the cardiac aortic sinus of AS mice.The experimental results showed that BYHWD could significantly reduce the plaque area of the whole aorta and the cardiac sinus in as mice,indicating that BYHWD had therapeutic effect on as mice.At the same time,BYHWD can reduce the lipid infiltration in the aortic sinus and the contents of TC,TG and LDL-C in the serum of as mice,so as to inhibit the progress of plaque and achieve the purpose of anti-AS.Foxp3,as a specific marker of Treg cells,plays an important role in the differentiation,development and immune regulation of Treg cells.It was found that BYHWD could significantly increase the number of Tregs,up regulate the expression of Foxp3,and increase the levels of TGF-βand IL-10 in serum.In addition,BYHWD can regulate TGF-β/Smad2 pathway,promote the differentiation of Treg cells and increase the number of Treg cells in AS mice.At the same time,the levels of IL-6,TNF-α,IFN-γ,MMP-9 in serum of AS mice decreased significantly after 4 weeks of BYHWD administration.It can be concluded that BYHWD can promote peripheral differentiation of Tregs,regulate inflammatory response and immune balance by regulating TGF-β/Smad2pathway,which is one of the potential mechanisms of its anti-as.Therefore,the next experiment needs to further verify the effect of BYHWD on the number and function of Treg cells in vitro.2.It was found that the effect of ox-LDL on the function of Treg cells might be realized by ox-LDL acting on Foxp3.CD4~+T cells were isolated by immunomagnetic beads in vitro.After stimulated by ox-LDL,BYHWD serum was given to intervene.The effect of BYHWD on peripheral differentiation of Treg cells based on the regulation of TGF-β/Smad pathway was detected in vitro.The results showed that BYHWD could promote the differentiation of Tregs in vitro,improve the immune imbalance of CD4~+T cells,regulate the expression of inflammatory factors,increase the level of anti-inflammatory factor TGF-βin the supernatant,and decrease the level of pro-inflammatory factors IL-6 and IFN-γ.In addition,BYHWD regulates Foxp3/TGF-β/Smad2 signaling pathway at the m RNA level.It can be concluded that BYHWD can regulate the number and function of Treg cells in vitro.3.A chitosan solution-based vortex-forced matrix solid phase dispersion method for the extraction and determination of four bioactive constituents from Ligustri Lucidi Fructus by high performance liquid chromatography:A simple and efficient sample preparation method to extract four bioactive compounds(echinacoside,specnuezhenide,oleuropein and nuezhenoside G13)from Ligustri Lucidi Fructus was established by vortex forced matrix solid phase dispersion(VFMSPD)method.Chitosan solution was applied as green eluent in this procedure and Celite AZO was employed as dispersant.High performance liquid chromatography(HPLC)equipped with ultraviolet(UV)detector was used to analyze the target analytes.The best result of the investigation was obtained with Celite AZO as dispersant,sample/dispersant ratio as 1:1,grinding for 2 min,1 m L high-viscosity chitosan solution(0.5 mg/m L)used as the elution reagent and vortex mixing for 1.5 min.The method exhibited a good linearity for the analytes(r~2>0.999).The absolute recoveries of the four target compounds in Ligustri Lucidi Fructus ranged from 90.7%to 98.8%and the relative recoveries of the target compounds ranged from 99.2%to 102%(RSD≤3.4%),which were obtained by the final optimization method.Consequently,the newly developed chitosan solution-based vortex-forced matrix solid phase dispersion(MSPD)combined with HPLC could be efficiently applied to extract and analyze the target compounds in Ligustri Lucidi Fructus samples. |
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