The Inhibitory Effect Of Chinese Materia Medica On Macrophage's Proliferation And Assay Of Its Proteomics With Different Expression.

1,ObjectiveDisease resulted from bacterial endotoxin is characterized by complex pathogenesis and widespread range, especially involving cardio-brain vascular diseases. The mechanism of Chinese materia medica to prevent and cure endotoxic shock maybe involves destroy of endotoxin, anti-inflammation, resistance of free radical, improvement of microcirculation, treatment of DIC, activation of reticuloendothelial system and protection of cell organs. Because of having the above characters, Chinese materia medica show better valuable for application in the clinic. So the study is major in whether Chinese herbs could improve/change the effect of endotoxin on expression of some proteins in monocytes/macrophages to adjust high levels or low levels of proteins expression at cellular level.To sieve the effective anti-endotoxic ingredients in heat-clearing and detoxicating herbs and blood-activating and stasis-removing (BASR) herbs, and discuss its mechanism at protein level, the inflammation cellular model induced by LPS was established, and then this model was applied to sieve the anti-endotoxic ingredients and analysis the proteomics. 2, Method and result 2.1 THP-1 cell line's property studying:THP-1 cell lines proliferation was determined by MTT method and WST-8 (CCK-8) kit, and the results indicated that THP-1 cell's proliferation induced by LPS achieved the peak after 24 hours, and the most suitable concentration ranged from 10μg/ml to 1μg/ml. Compared with control group, there were significantly difference between control group and LPS group (p<0.01) , but there were no difference between the two concentrations. Inaddition to LPS group, PHA and ConA can also induce THP-1 cell's proliferation, and the peak hour was same to LPS group's. The most suitable concentration of LPS was 5ug/ml in both PHA and ConA groups, which had significantly difference to control group (p<0.01) . After THP-1 cells matured by PMA, experiment of phagocytizing CRBC showed that all three stimulants can activate matured THP-1 cells' phagotrophic function, but LPS group's function was stronger than others, and made significantly difference to control group.2.2 Establishment of THP-1 cellular inflammation model induced by LPS: ?WST-8 method was adopted in experiment of proliferation, results indicated that 1 ng/ml was the optimal concentration of LPS to induce THP-1 cells proliferation, moreover, cells proliferation reach the peak after 24 hours, and they maintained 48 hours, descended after 72 hours.(2) ELISA kit was applied to detecting TNF-aand IL-lp\The secretion of cytokines (TNF-a,IL-l(3) reached the peak after 12 hours, and made significantly difference to control group (/?<0.01). Above data showed that THP-1 cellular model of inflammation induced by LPS is successfully established which made foundation for the drug screening.2.3 Screening the effective ingredients of Chinese materia medica:20 monomers constituents made from Chinese materia medica were sieved through the cellular model induced by LPS. Results showed that Total Tanshinone, Tanshinone I ,Tanshinone IIA and Tanshinonin can not only obviously inhibit cell proliferation of model group ranged from lOug/ml to lug/ml, and also can inhibit TNF-a and IL-iPproduction of model group.2.4 The influence of Effective ingredients in Chinese drugs on the proteomics of cellular model induced by LPS:The effective ingredients sieved out from model were studied by two- dimensional electrophroesis to identify proteomics. The results indicated that there were 31 protein spots in different expression in every group. Total Tanshinone, Tanshinone I ,Tanshinone II A, Emodin and Berberine have two-ways-regulation influence on proteins of model group. Above data demonstrated that Chinese drugs regulated total proteins in cells. Following we need to use MALDI-TOF-MS to identify structure and function of proteins.2.5 Applying SELDI-TOF-MS to assay proteome with different expression:IMAC3 and WCX2 arrays were used to study proteome in THP-1 cells with different treatment. The results indicated that normal THP-1 cell group, model group and Chinese drugs group respectively had different icon of protein expressions in IMAC3 and WCX2 arrays. There were 65 expressed protein peaks in model group and control group icon in 1MAC3 array, in which there were 11 proteins with different expression(account28.9%);InWCX2 array there were 87 expressed protein peaks in both group, 14 proteins with different expression(account27.4 % ).In addition, 12 protein peaks were captured in common by both IMAC3 and WCX2 arrays, which indicate that there was 23.5% protein peak in WCX2 arrays can be captured by IMAC3 arrays, 31.6% protein peak in IMAC3 arrays can be captured by WCX2 arrays. Combining the protein peak displayed in two icon, there were about 24.7% peak possessed in common. Comparing two arrays of capturing protein peak, we know that WCX2 arrays capture more protein peak than IMAC3 arrays, but there were no difference between respective captured proteins in different expression. According to CVS data came from proteins with different expression in various compared groups, we concluded that every medicine group can regulate proteins with different expression to some extent. Meanwhile, 3 pairs of proteins (5.7KDa, 10.7KDa, 10.9KDa) were found in course of analyzing phosphorylation and dephosphorylation in two arrays. Some protein had two peaks, needing further analysis. The above proteins in different expression need further identify and analyze binding database to gain concerned proteins of this experiment. 3% Conclusion:3.1 THP-1 cell can be selected as the stimulate cell of inflammatory cellar model and be transformed to endotoxic cellular model after induced by LPS.3.2 The endotoxic cellular model can became platform for Chinese drugs screening in laboratory.3.3 Various effective ingredients had different influence on proteomics of cellular model induced by LPS. According to analysis of proteins with different expression, molecular weight of proteins targeted by Chinese drugs ranged from 6Kda to 129Kda, and isoelectric point was from pH4 to pH7, which made way to mass-spectrum identification.3.4 Using SELDI-TOF-MS assay proteins with different expression:3.4.1 There were 14 proteins with different expression in WCX2 array and 11 in IMAC3 array. From expressed protein peaks in total icons, captured proteins in WCX2 array were more than those in 1MAC3 array, but each other made no difference in protein with different expression capture.3.4. 2 12 protein peaks were captured by IMAC3 and WCX2 arrays, and every medicine group can down-regulate or up-regulate them at different extents.3.4.3 3 pairs of protein(5.7KDa,10.7KDa,10.9KDa) were found in course of analyzing phosphorylation and dephosphorylation, but they need further indentification.3.4.4 We used two-dimensional electrophoresis technique(2-DE)and SELDI to capture proteins ranged from 2.5KDa to 30KDa in common in spite of different technique, andthere were 13 approximate matching-proteins after considerable bolting, among which 17KDa and 15KDa may be the significant proteins needing further identification. 3.4. 5 From 13 proteins with different expression expressed in common, we would find the correlative protein in the process of endotoxin induceing inflammation binding analysis of data base.