Effect Of Biological Active Constituents From Echinacea Purpurea L. On Inflammation Of Vascular Smooth Muscle Cells In Rats And Relevant Mechanism

Vascular smooth muscle is the main component of the vessel wall, and itsinflammation can lead to smooth muscle cells proliferation and migration, resulting invascular lesions of atherosclerosis and vascular stenosis after operation. Echinaceapurpurea L. as a main medicinal species of Echinacea, can enhance cellular immunityand treat a variety of inflammation and infection. In this paper, the primary culture ofrat vascular smooth muscle cells and identification were studied; the essential oilsfrom E. purpurea and cichoric acid effects on inflammation of rat vascular smoothmuscle cells and the influence of the mechanism cause by lipopolysaccharide weretested. The results are as follows:1. Rat vascular smooth muscle cell line was set up. The Cells were isolated byenzyme digestion method, the smooth muscle cells were purified by the natural purificationmethod, the results showed the cells presented a typical "Gu Feng" growth. Identified thefifth generation cells by immunohistochemistry staining, we found the vascular smoothmuscle cell line was established successfully by the positive expression of alphasmooth muscle actin in the cytoplasm.2. We studied on the effects of the essential oil and cichoric acid from E. purpurea onthe growth of vascular smooth muscle cell, and their medicinal concentration rangewere determined. MTT method was used to detect the effects of essential oil andcichoric acid on proliferation of vascular smooth muscle cells. The results show thatthere was no inhibitory effect on cell growth when the essential oil’s concentration islower than0.01%; it had no inhibitory effect on cell growth when the cichoric acid’sconcentration is less than15μg/ml.3. The inflammation models of vascular smooth muscle cells were established whichwere induced by lipopolysaccharide. We studied on the optimum concentration andtime of inflammation of vascular smooth muscle cells stimulated by thelipopolysaccharide, and detected the expression of IL-6mRNA by real-timefluorescence quantitative PCR. The optimum stimulation concentration of LPS was5μg/ml, and the most suitable time was1h.4. The expression of mRNA cytokine was detected by real-time fluorescencequantitative PCR. The effects of the essential oil and cichoric acid on vascular smoothmuscle cell inflammation were studied. Compared with the model group, when theessential oil concentration was0.005%, the expression of TNF-α mRNA was decreased by66.5%, the IL-6mRNA expression was decreased by37.0%, the COX-2mRNA expression was decreased by35.5%; when the essential oil concentration was0.01%, the expression of TNF-α mRNA was decreased by77.3%, the IL-6mRNAexpression was decreased by52.6%, the COX-2mRNA expression was decreased by40.8%; when cichoric acid concentration was5μg/ml, the expression of TNF-αmRNA were decreased by56.9%, the IL-6mRNA expression was decreased by34.4%, the COX-2mRNA expression was decreased by29.0%; when the cichoricacid concentration was15μg/ml, the expression of TNF-α mRNA was decreased by23.1%, the IL-6mRNA expression was decreased by29.5%, the COX-2mRNAexpression was decreased by46.6%. The essential oil and cichoric acid from E.purpurea can down-regulate the expression of pro-inflammatory cytokines mRNAand inhibit inflammatory reaction.5. The differential expression of NF-κ B/p65, p38/MAPK protein by western blotwere detected to explore the anti-inflammatory mechanism of essential oil andcichoric acid on the vascular smooth muscle cells. Compared with the model group,when the essential oil was0.005%, the expression of NF-κb/p65protein wasdecreased by19.5%, the expression of p38/MAPK protein was decreased by14.0%;when the essential oil was0.01%, the expression of NF-κb/p65protein was decreasedby30.3%, the expression of p38/MAPK protein was decreased by19.8%; when thecichoric acid concentration was5μg/ml, the expression of NF-κb/p65protein wasdecreased by30.3%, the expression of p38/MAPK protein was decreased by11.9%;when the cichoric acid concentration was15μg/ml, the expression of NF-κb/p65protein was decreased by39.6%, the expression of p38/MAPK protein was decreasedby13.0%. The essential oil and cichoric acid from E. purpurea can down-regulateexpression of NF-κB/p65, p38/MAPK, and influence their ability of entering tonucleus and combining the corresponding target sequence, then exert theanti-inflammatory effects.These results can provide a theoretical basis for further development andutilization of E. purpurea.