| Object:In this study,lipopolysaccharide(LPS)was used to induce human umbilical vein endothelial cells(HUVECs)to establish an inflammatory model.Molecular biology and cell biology were used to verify the correlation between the inflammatory response of model cells and 5-HT signaling pathway in vitro.At the same time,the regulatory effect of serum containing Yiqi Jianpi Recipe on the inflammatory response of model cells was observed and its mechanism was discussed.Further enrich the treatment of atherosclerosis(AS)from the spleen,in order to provide reference and reference for targeted treatment of cardiovascular diseases in traditional Chinese medicine.Material and method:1.Preparation of Serum24 SPF male 8-week-old SD rats were randomly divided into four groups: normal group,model group,Yiqi Jianpi Preparation group and atorvastatin group.Normal group and model group were given normal saline by gavage.The latter two groups were given Yiqi Jianpi Preparation and atorvastatin by gavage once a day for 7 days.After 2 hours of the last administration,blood was collected from the abdominal aorta,centrifuged,supernatant was collected,and drug-containing serum of the same group was merged.Sterile filtration was carried out with a 0.22 ? m disposable filter and stored in a refrigerator at-80 ? for reserve.2.Effect of Yiqi Jianpi Preparation on LPS-induced inflammation in HUVECs Human umbilical vein endothelial cells(HUVECs)cultured in vitro were randomly divided into four groups: normal group,model group,Yiqi Jianpi Preparation group and atorvastatin group.Except the normal group,the other groups were stimulated with LPS(1.5ug/ml)for 24 hours.At the same time,normal serum was used in normal group and model group,and corresponding drug serum was used in treatment group to intervene cells.The intervention was carried out at three time points: 2h,4h and 8h(adding drugs at 22 h,20h and16 h respectively).Cells were collected after 24 h.The expression of cytokines IL-6,TNF-?and 5-HT in each group was detected and analyzed by ELISA.3.Study on the mechanism of Yiqi Jianpi Preparation improving inflammation level of HUVECs induced by LPS Human umbilical vein endothelial cells(HUVECs)cultured in vitro were randomly divided into six groups:normal group,model group,antagonist group,Yiqi Jianpi Preparation group,Yiqi Jianpi Preparation + antagonist group and atorvastatin group.Except the normal group,the other groups were stimulated with LPS(1.5 ug/ml)for 24 hours.At the same time,normal serum,normal serum + antagonist,serum of Yiqi Jianpi Preparation,serum of Yiqi Jianpi Preparation + antagonist and atorvastatin serum were used to intervene for 8 hours(after 16 hours),and cells were collected after 24 hours.Western-blot was used to detect the expression of PKC,p-PKC and NF-?B.Quantitative real-time Polymerase Chain Reaction(Real-time PCR)was used to detect the expression of PKC and NF-?B.Results:1.The expression of interleukin-6(IL-6)in HUVECs Compared with the normal group,the expression of IL-6 increased in the model group(P< 0.05);compared with the model group,the expression of IL-6 in the Yiqi Jianpi Preparation group and atorvastatin group decreased(P < 0.05 or P < 0.01).In the experiment,three time echelons(2h,4h,8h)of Yiqi Jianpi Preparation were used to intervene the model cells.Compared with the model group,the effect of 8h was the best by statistical analysis.2.The expression of Tumor Necrosis Factor-alpha(TNF-?)in HUVECs Compared with the normal group,the expression of TNF-? increased in the model group(P < 0.05);compared with the model group,the expression of TNF-? in the Yiqi Jianpi Preparation group and atorvastatin group decreased(P < 0.05 or P < 0.01).In the experiment,three time echelons(2h,4h,8h)of Yiqi Jianpi Preparation were used to intervene the model cells.Compared with the model group,the effect of 8h was the best by statistical analysis.Compared with atorvastatin group,there was no significant difference in TNF-? expression between Yiqi Jianpi Preparation group and at orvastatin group(P > 0.05).3.The expression of 5-HT in HUVECs Compared with the normal group,the expression of 5-HT increased in the model group(P < 0.05);compared with the model group,the expression of 5-HT decreased in the Yiqi Jianpi Preparation group and atorvastatin group(P < 0.05 or P < 0.01).In the experiment,three time echelons(2h,4h,8h)of Yiqi Jianpi Preparation were used to intervene the model cells.Compared with the model group,the effect of 8h was the best by statistical analysis.Compared with atorvastatin group,there was no significant difference in 5-HT expression between Yiqi Jianpi Preparation group and atorva statin group(P > 0.05).4.The expression of protein kinase C(PKC)in HUVECs Compared with the normal group,the expression of PKC protein and mRNA in the model group increased significantly(P < 0.01);compared with the model group,the expression of PKC protein and mRNA in the antagonist group decreased significantly(P < 0.05),while that in the other treatment groups decreased significantly(P < 0.01);compared with the antagonist group,the expression of PKC protein and mRNA in the Yiqi Jianpi Preparation group,Yiqi Jianpi Preparation + antagonist group were sign ificantly higher(P < 0.01).There was a significant decrease(P < 0.01).In summary,the therapeutic effects of each treatment group were Yiqi Jianpi Preparation + Antago nist Group > Atorvastatin Group > Yiqi Jianpi Preparation Group > Antagonist Group.5.The expression of phosphorylated protein kinase C(p-PKC)in HUVECs Compared with the normal group,the expression of p-PKC protein in the model group increased significantly(P < 0.01);compared with the model group,the expression of p-PKC protein in each treatment group decreased significantly(P < 0.01);comp ared with the antagonist group,the expression of p-PKC protein in the Yiqi Jianpi Preparation group and the Yiqi Jianpi Preparation + antagonist group decreased significantly(P < 0.01).In summary,the therapeutic effects of each treatment group were Yiqi Jianpi Preparation + Antagonist Group > Atorvastatin Group > Yiqi Jianpi Preparation Group > Antagonist Group.6.The expression of nuclear factor ?B(NF-?B)in HUVECs Compared with the normal group,the expression of NF-?B protein and mRNA in the model group increased significantly(P < 0.01);compared with the model group,the expression of NF-?B protein and mRNA in the antagonist group decreased significantly(P < 0.01 or P < 0.05),while the expression of NF-?B protein and mRNA in the other treatment groups decreased significantly(P < 0.01);Compared with the antagonist group,the expression of NF-?B protein and mRNA in Yiqi Jianpi Preparation group and Yiqi Jianpi Preparation + antagonist group decreased significantly(P <0.01).In summary,the therapeutic effects of each treatment group were Yiqi Jianpi Preparation + Antagonist Group > Atorvastatin Group > Yiqi Jianpi Preparation Group >Antagonist Group.Conclusion:1.The up-regulation of pro-inflammatory cytokines IL-6 and TNF-? in LPS-induced HUVECs and the increase of 5-HT secretion in all groups of inflammatory model cells suggest that the over-secretion of 5-HT and endothelial inflammatory reaction may be the mechanism of promoting the development of AS.2.Yiqi Jianpi Preparation(Preparation of Yiqi Jianpi Method)can regulate the immune inflammation process of atherosclerosis by targeting 5-HT2 A receptor,improve the secretion of inflammatory factors,and inhibit the activation of 5-HT and its downstream PKC-NF-?B signaling pathway,which may be an important mechanism target for the intervention of early AS by Yiqi Jianpi Preparation.3.Both Yiqi Jianpi Preparation and 5-HT2 A Receptor Antagonist can improve the inflammatory response of model cells.The former is better than the latter,suggesting that Yiqi Jianpi Preparation may play an important role in improving the inflammatory response of endothelial cells on the basis of further antagonizing the 5-HT signal transduction pathway,thereby interfering with the occurrence and development of early AS. |
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