| BackgroundAtherosclerosis（AS）is a chronic inflammatory disease occurring in the arterial wall.Inflammatory reaction plays an important driving role in the development of AS.Plaque rupture or secondary thrombosis is closely related to clinical acute ischemic events of AS.Inflammatory response plays an important role in the formation and progression of vulnerable plaque.Inhibiton of inflammatory response to enhance plaque stability and delay the progression of vulnerable plaques.Si-Miao-Yong-An decoction is a representative prescription for removing heat and detoxifying toxins and promoting blood circulation.The team’s preliminary study found that Si-Miao-Yong-An decoction can stabilize vulnerable plaques by regulating inflammation,oxidative stress extracellular matrix.Whether Si-Miao-Yong-An decoction can improve plaques stability by regulating inflammasome activation of（nucleotide-binding oligomerization domain,leucine rich repeat and pyrin domain containing 3,NLRP3）,and the mechanism of inflammasome activation is worth further discussion.PurposeThis study,apolipoprotein E knockout(Apo E-/-)mice fed with western diet（WD）to establish AS model,Si-Miao-Yong-An decoction was used as an intervention to evaluate its effect on aortic pathological changes and plaque stability in AS mice,and to explore the mechanism of Si-Miao-Yong-An decoction in regulating the activation of NLRP3 inflammasome,so as to provide experimental basis for clinical application of Si-Miao-Yong-An decoction.Method1.The experimental animals were fed for 1 week.8 C57BL/6J wild type mice were used as normal control group,and 40 Apo E-/-mice were randomly divided into model group,Atorvastatin group,Si-Miao-Yong-An decoction low dose group,Si-Miao-Yong-An decoction medium dose group and Si-Miao-Yong-An decoction high dose group,with 8 mice in each group.The control group was given normal diet,and the other groups were given“WD”for 8 weeks.After the establishment of AS model,the mice were given drugs for 8 weeks.All the mice in each group were taken at the end of the 16th week of the experiment;2.After sampling,HE staining was performed on paraffin section of aortic sinus to calculate the ratio of inner and middle membrane area and the thickness of fibrous cap,and Movat staining was used to calculate the foam cells content,collagen content and elastin content in the plaque;3.Serum IL-18 and IL-1βwere detected by ELISA;4.The expressions of NLRP3,caspase-1,IL-18 and IL-1βin paraffin section of aortic sinus were detected by immunohistochemistry;5.The expression of GSDMD-N protein related to pyroptosis was detected by Western-blot.6.SOD activity and MDA content were detected in aortic tissue homogenate;7.The protein expressions of Nrf2,Keap1,Trx1 and TXNIP in aortic tissues were detected by immunohistochemistry.Result1.Pathological changes and plaque stability evaluation of mouse aortaThe results of HE staining showed that compared with the control group,the ratio of inner and middle membrane area increased and the thickness of fibrous cap increased in model group（P<0.01）.Compared with model group,the ratio of inner and middle membrane area in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups decreased（P<0.01）,and the thickness of fibrous cap in Si-Miao-Yong-An decoction medium dose and high dose groups increased（P<0.01）.Compared with Atorvastatin group,there was no statistical significance in the ratio of inner and middle membrane area and the thickness of fibrous cap in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups（P>0.05）.Movat staining results showed that compared with the control group,the foam cells content,collagen content,elastin content in model group were increased（P<0.01）;Compared with model group,the content of foam cells in plaques in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups was decreased（P<0.05 or P<0.01）,the content of collagen in plaques in Si-Miao-Yong-An decoction high dose group was increased（P<0.05）,and the content of elastin in plaques in Si-Miao-Yong-An decoction low dose group was increased（P<0.01）.Compared with Atorvastatin group,there were no statistical differences in foam cells content and collagen content in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups（P>0.05）,and there were no statistical differences in elastin content between Si-Miao-Yong-An decoction medium dose and high dose groups（P>0.05）.2.Evaluation of NLRP3 inflammasome activation in mouse aortaELISA results showed that compared with the control group,the expression level of inflammasome cytokine IL-18 was increased in the model group,with statistical significance（P<0.01）.Compared with model group,the expression of IL-18 in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups decreased（P<0.01）;Compared with Atorvastatin group,the expression of IL-18 in Si-Miao-Yong-An decoction medium dose group was not statistically significant（P>0.05）.Compared with the control group,the expression of IL-1βin model group was increased,with statistical significance（P<0.05）.Compared with model group,the expression of IL-1βin Si-Miao-Yong-An decoction medium dose and high dose groups decreased,with statistical significance（P<0.05 or P<0.01）,and the inhibitory effect was more obvious in Si-Miao-Yong-An decoction high dose group.Compared with Atorvastatin group,the expression of IL-1βin Si-Miao-Yong-An decoction low dose,medium dose and high dose groups had no statistical significance（P>0.05）.Compared with the control group,the expression levels of NLRP3,Caspase-1,IL-18and IL-1βin model group were increased（P<0.05 or P<0.01）.Compared with model group,the expressions of NLRP3,caspase-1,IL-18 and IL-1βin Si-Miao-Yong-An decoction low dose,medium dose and high dose groups were decreased（P<0.05 or P<0.01）.Compared with Atorvastatin group,the expression of NLRP3 protein in Si-Miao-Yong-An decoction medium and high dose groups was not statistically significant（P>0.05）,the expression of caspase-1 and IL-18 protein in Si-Miao-Yong-An decoction low,medium and high dose groups was not statistically significant（P>0.05）,and the expression of IL-1βin Si-Miao-Yong-An decoction low dose group was not statistically significant（P>0.05）.Western-blot analysis showed that compared with the control group,the protein expression level of GSDMD-N in model group was increased（P<0.05）.Compared with model group,the protein expression level of GSDMD-N in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups was decreased（P<0.05 or P<0.01）.3.Evaluation of Nrf2/Trx1/TXNIP signaling pathwaysThe results of SOD activity and MDA content in aorta tissue homogenate showed that compared with the control group,SOD activity in model group decreased and MDA content increased（P<0.05 or P<0.01）.Compared with model group,SOD activity in Si-Miao-Yong-An decoction high dose group was increased（P<0.05）,while MDA content in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups was decreased（P<0.05 or P<0.01）.Compared with Atorvastatin group,there were no significant differences in SOD activity and MDA content between Si-Miao-Yong-An decoction low dose,medium dose and high dose groups（P>0.05）.Immune histochemical method group observed node protein Keap1,Nrf2 signaling pathways,Trx1,TXNIP protein expression of the study showed that compared with model group,Si-Miao-Yong-An decoction intervention group had no obvious effect on Keap1 protein expression,Si-Miao-Yong-An decoction and high dose group can increase the Nrf2protein expression（P<0.05 or P<0.01）,Si-Miao-Yong-An decoction high dose group can increase Trx1 protein expression（P<0.05）,Si-Miao-Yong-An decoction TXNIP dose group decreased,protein expression（P<0.01）.Compared with Atorvastatin group,the protein expressions of KEAP1,Nrf2,Trx1 and TXNIP in Si-Miao-Yong-An decoction low dose,medium dose and high dose groups were not statistically significant（P>0.05）.Conclusion1.Si-Miao-Yong-An decoction can improve the degree of intimal hyperplasia of Apo E-/-mice aorta,increase the thickness of plaque fibrous cap,reduce the formation of foam cells in plaque,increase the content of collagen and elastin and increase the stability of plaque.2.Si-Miao-Yong-An decoction inhibited inflammasome activation by reducing protein expression related to the inflammasome in mice aorta.3.Si-Miao-Yong-An decoction inhibited the activation of NLRP3 inflammasome and was related to the NRF2/Trx1/TXNIP signaling pathway. |
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