| ObjectiveBased on the theory of inflammation resolution,this project is to study the efficacy of Shenlian（SL）extract on ox-LDL-induced macrophage-derived foam cell formation and its apoptosis and comparison of its fractions.There are the objective that further studing the mechanism of SL extract regulating the apoptosis of foam cells by endoplasmic reticulum stress.On the one hand,there is need to clear that the efficacy of SL extract on the process of AS inflammation resolution and the remodeling of lipid peroxidation inflammatory injury balance.On the other hand,by evaluating and comparing the differences in the efficacy and activity of different components of SL extract,it provides preliminary research points and ideas for the scientific and superior efficacy of SL extract.At the same time,it provides a new theoretical basis and experimental basis to treat atherosclerosis through SL extract,and reveals the pharmacological mechanism of endoplasmic reticulum stress during lipid uptake.Methods1.The mechanism of SL extract on plaque lipid clearance in AS model miceThe atherosclerosis mice model was established by right carotid cannula combined with high-fat feeding in ApoE knockout C57BL/6J mice.ApoE knockout mice were randomly divided into 6 groups according to their body weight,which were sham-operation group（Sham）,model control group（Model）,positive drug rosuvastatin group（P,0.1 mL/10g/d）,the low dose of SL extract group（SL-L,95 mg/kg）/d),the medium dose of SL extract group（SL-M,190 mg/kg/d）and the high dose of SL extract group（SL-H,380 mg/kg/d）.Animals were dissected at the end of the 9th week.The expression of phagocytic receptor CD36 in the endothelium of aortic arch was detected by immunohistochemistry.The expression of CD36 in the subarachnoid macrophage was detected by immunofluorescence double staining technique.The immunohistochemical technology was used to detect the protein expression of CHOP and XBP-1 in the aortic arch stress-promoting signaling pathway in AS model mice.The AS model.The protein expression of GRP-78 which the endoplasmic reticulum stress marker was detected by immunohistochemistry.2.Efficacies identification of SL extract and its components accelerate the lipid clearance of ASPeritoneal macrophages were induced by ox-LDL to establish an inflammatory injury model.Peritoneal macrophages were collected from the C57BL/6J mice.After culturing for 72 hours,the peritoneal macrophages were randomly divided into 7 groups:negative control group（NC）,model group(Model,20 mg·L-1 ox-LDL),water-soluble extract of Salvia miltiorrhiza group(4.32 mg·L-1 DS+20 mg·L-1 ox-LDL),Liposoluble extract of Salvia miltiorrhiza group(2.5 mg·L-1 DZ+20 mg·L-1 ox-LDL),Liposoluble extract of Andrographis paniculata group(3.18 mg·L-1 C+20 mg·L-1 ox-LDL),and the Shenlian extract group(10 mg·L-1 SL+20 mg·L-1 ox-LDL),endoplasmic reticulum stress inducer group(TM,1 g·L-1).After treatment for 24 hours,the accumulation of lipids were stained of Oil red oxygen.,and the apoptosis of peritoneal macrophages was detected by Annexin V-FITC/PI staining.3.The mechanism of SL extract and its components accelerate the lipid clearance of AS Peritoneal macrophages were induced by ox-LDL to establish an inflammatory injury model.Groups are as above to methods 2.The expression of phagocytic receptor CD36and Annexin A1 mRNA was detected by RT-PCR.The expression of endoplasmic reticulum stress C/EBP homologous protein CHOP mRNA was detected by RT-PCR.The expression of scavenger receptor CD36 were detected by indirect immunofluorescence assay.The expression of intracellular calcium concentration were detected by flow cytometry.The expression of CHOP、Caspase-12、XBP-1 were detected by Western blot.The Caspase-3 activity was measured by chromatometry.Results1.The mechanism of SL extract on plaque lipid clearance in AS model miceCompared with the Sham group,the expression of CD36 protein in the aortic arch of the model group was up-regulated,and the expression of CHOP protein was up-regulated which is involved in the apoptosis signaling pathway in the endoplasmic reticulum stress.The expression level of XBP-1 protein,a key regulator of the survival signal pathway,was down-regulated,and the expression of endoplasmic reticulum stress marker GRP-78was up-regulated.Compared with the model group,by treated with rosuvastatin,the expression of CD36 protein was up-regulated,and the expression level of CHOP protein was down-regulated.The expression level of XBP-1 protein was up-regulated,and the expression of GRP-78 protein was down-regulated.Compared with the model group,the expression of CD36 protein in the aortic arch was up-regulated in each dose group of SL extract,and the expression level of CHOP protein was down-regulated.The expression level of XBP-1 protein was up-regulated,and the expression of GRP-78 protein was down-regulated.2.Efficacies identification of Shenlian extract and its components accelerate the lipid clearance of ASThe results of oil red O staining in peritoneal macrophages showed that SL extract could significantly reduce lipid deposition in macrophages,and the lipid-soluble extract of Andrographis paniculata was more effective;The results of Annexin V-FITC/PI staining showed that SL extract could significantly reduce the apoptosis of macrophages,and the extract of the salvia miltiorrhiza extract was more effective（P<0.05）.3.The mechanism of Shenlian extract and its components accelerate the lipid clearance of ASCompared with the negative control group,the model group can up-regulate the expression of CD36 mRNA and protein levels,and increase the expression of CHOP protein levels and active the cleaved of Caspase-12 protein.The experimental results of the endoplasmic reticulum stress inducing agent tunicamycin group were consistent with the model group.Compared with the model group,based on the up-regulation of CD36expression in the model group,SL extract can further up-regulate the expression of CD36mRNA and protein levels in macrophage phagocytic receptors;while SL extract can effectively reduce the phagocytosis of ox-LDL.Calcium ion concentration in macrophages significantly reduced the expression levels of CHOP and Caspase-12.On the basis of statistically significant differences in the efficacy of the model group and the effectiveness of the whole prescription,the liposoluble extract of Andrographis paniculata was superior to the extract of Salvia miltiorrhiza（P<0.05）.The extract of Salvia miltiorrhiza reduced the expression levels of CHOP,Caspase-12 and Caspase-3was superior to the liposoluble extract of Andrographis paniculata（P<0.05）.Conclution1.SL extract could enhance the expression of phagocytic receptor CD36 in the aortic arch of AS model mice,promote the phagocytosis of lipid peroxide,down-regulate the expression of CHOP,up-regulate the expression of XBP-1,and reduce the expression of the ER stress marker GRP-78,thereby relieves endoplasmic reticulum stress,fuither improves the efficacy of AS plaque lipid clearance,maintains effective burial effects,and promotes inflammation resolution.2.SL extract which can significantly up-regulate the expression of CD36 in peritoneal macrophages,promote the uptake of ox-LDL,and effectively reduce the accumulation of lipids and apoptosis in peritoneal macrophages after ox-LDL uptake,which improves the state of macrophage survival,thereby effectively eliminating lipid peroxides.3.SL extract may inhibit lipid peroxide-induced apoptosis through the endoplasmic reticulum CHOP/Caspase-12/Caspase-3 pro-apoptotic signaling pathway.4.The liposoluble extract of Andrographis paniculata was superior to the extract of Salvia miltiorrhiza.The extract of Salvia miltiorrhiza reduced the expression levels of CHOP and Caspase-12 was superior to the liposoluble extract of Andrographis paniculata. |
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