| Objective:Atherosclerosis (AS) is a common pathological basis of cardiovascular and cerebrovascular diseases.It has long been associated with acute cardiovascular events and harms to human healtlth seriously.This research studies the effects and mechanism of Huanglian Jiedu Decoction(HLJDD) on AS by in vivo and in vitro experiments,based on the polarization and inflammatory effects of M1/M2macrophages.It can accumulate scientific evidence for understanding the "evil" theory of AS of traditional Chinese medicine based on the inflammatory reaction, and provide the experimental basis for the further research on the effect and mechanism of heat-clearing and detoxifying drugs such as HLJDD on AS based on the inflammatory regulating.Methods:1.In vivo studies:1)The atherosclerotic model of rabbits was established. After6weeks, Model groups were respectively treated with different dose HLJDD.At the end of the6,10,14weeks, the serum of model animals was collected to measure blood fat which was the important driving factor of AS, C-reactive protein(CRP) which was the most representative inflammatory marker of AS, and TNF-α(M1stimulating factor and active product) and TGF-β1(M2stimulating factor and active product). The intervention effects of HLJDD were analyzed.2)At the end of the14week,the aortic tissues of model rabbits were obtained to detect the intervention effects of HLJDD on AS local pathological damages and the distribution of the M1/M2in damage position in experimental animals.2.1n vitro studies:1)The preparation of Cells and medicated serums:The C57BL/6mice bone marrow macrophages were induced by M-CSF and cell purity was determined using flow cytometry. OX-LDL induced foam cells derived macrophage, the inductive effect was identified by oil-red O staining. The HLJDD medicated serums were prepared with C57BL/6mice. MTT method screened sample concentration which had no obvious impact on the number of macrophage and foam cell.2)Detecting the influences of medicated serums on Mφ and foam cell differentiation and releasing inflammatory factors:(l)The intervention effects of HLJDD medicated serums on Mcp and foam cell polarization were detected by Flow cytometry.(2)The influences of HLJDD medicated serums on the release of TNF-aand TGF-β1were checked with ELISA method.3)Detecting signaling pathways which was closely related to macrophage inflammation regulation:(1) The influences of HLJDD medicated serums on TLR4and NF-kB mRNA expression level were tested with RT-PCR method;(2) The influences of HLJDD medicated serums on TLR4and NF-kB protein expression level were tested with Western blot.Results:1.In vivo studies:1) Serum detection:(1)Blood lipids:In the AS forming process, the TC, TG, LDL levels of model rabbits in blood on the whole showed a trend of rise(P<0.05or P<0.01or P＜0.001). Each dose group of HLJDD had no significant intervention effect on them(P>0.05). The HDL level in blood increased in early (6weeks ago)(P＜0.001) and late was steady (6-14weeks)(P>0.05). Each dose group of HLJDD could improve the HDL level of model animals (P<0.05or P＜0.001).(2)CRP: In the AS forming process, the CRP level of model rabbits in blood showed a trend of sharp rise in the overall(P＜0.01or P＜0.001), different doses of HLJDD could decrease the CRP level of model animals (P＜0.05or P＜0.05or P＜0.001).(3)TNF-a:In the AS forming process, the TNF-alevel of model rabbits in blood showed a trend of massive rise (P＜0.001), different doses of HLJDD had significant inhibitory effect on the TNF-alevel of experimental animals (P＜0.05or P＜0.05or P＜0.001).(4)TGF-(31:in the AS forming process, the TGF-β1level of model rabbits assumed the overall downward trend (P＜0.001), the TGF-β1level significantly increased in different HLJDD dose groups(P＜0.05or P＜0.05or P＜0.001), especially in the10th weeks or so.2)Organization test:(1)The macroscopic and microscopic pathological examination:in14weeks, the AS damages of model rabbits were apparent. The large, middle doses of HLJDD had obvious relief effects on the atherosclerotic process and damage.(2)The inimunohistochemical detection of the distribution of M1/M2in the AS plaques:in14weeks, the CCR7+M1cells mainly distributed in central plaques in the model control group, the CD206+M2cells mainly distributed on the edge of the plaques. The CD206+M2cells extended to the whole patches and the CCR7+MI cells decreased significantly in the large dose group, compared with the model control group, the CD206+M2cells distributed more widely in the middle and small dose groups.2.1n vitro studies:1) Cells and medicated serums:After being induced by M-CSF, the purity of macrophage derived from C57BL/6mice bone marrow was up to94.37%. The morphology of the foam cells induced by ox-LDL was typical and used as the experimental research on cells. The result determined by MTT method showed that the medicated serums had no obvious impact on the number of test cells in final concentration of20%.2)The influences of HLJDD medicated serums on Mcpand foam cell differentiation and releasing inflammatory factors:(1) The intervention effects on Mcp and the foam cells polarization:under the intervention of large, middle and small dose of HLJDD medicated serums:the CD16/32molecular positive proportions of macrophage(12.1%,17.8%,13.3%) were close to that of normal control group (11.7%), the CD206molecular positive proportion of large dose which was highest was3.1times of the normal control group (11.7%);Compared with normal control group (the proportion of CD16/32was46.9%, that of CD206was21.4%), the CD16/32(34.8%,37.1%,49.6%) and CD206(27.3%,19.9%,20.2%) molecular positive proportions showed a two-way volatility (but the downward trend of CD16/32and the rising trend of CD206were more obvious).(2) The influences on the release of TNF-aand TGF-β1:compared with normal control group, medicated serums could stimulate Mcp to secrete TNF-a(P<0.05) and TGF-β1(P<0.001), but the latter one was more obvious; For the macrophage foam cells, some dose of medicated serum could reduce the secretion of TNF-a(P<0.05), and promote the release of TGF-β1(P<0.05or P<0.001).3)The effects of HLJDD medicated serums on the expression of M9inflammation-related signal pathways:Compared with the normal control group serum, different doses medicated serum had different degrees of inhibition on TLR4and NF-kB mRNA/protein expression levels of macrophage (P＜0.05or P＜0.01or P<0.001), and with a dose dependent(P<0.05or P＜0.001).Conclusion:l.In vivo studies:Along with the progress of AS,the contents of blood lipid disorder seriously,the levels of TNF-aelevate and those of TGF-β1reduce, the inflammatory reaction using CRP as an indication enhances,and some dose of HLJDD has restriction on the development of inflammation and can regulate the release of inflammatory factors.meanwhile, HLJDD with a certain dose can delay or reduce the degree of AS damage. Its mechanism may be related to the promoting effects on M2differentiation and the limiting effects on M1differentiation in local tissue of AS.2.1n vitro studies:For Mφ,HLJDD medicated serums can promote the differentiation of Mφ to M2and the secretion of TGF-β1.For foam cells derived from macrophage,HLJDD can regulate bidirectionally cell polarization, inhibit TNF-α and promote TGF-β1. These may be the important reasons that HLJDD regulates the release of inflammatory cytokines,inhibits inflammation and intervenes the local distribution of M1/M2in the process of AS. HLJDD medicated serums with a certain dose can inhibit the expressions of TLR4and NF-kB which are the signaling pathways associated with the regulation of inflammation. This may be one of the important mechanisms that HLJDD intervenes the inflammatory responses of AS based on macrophages in vivo and vitro. |
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