Study On The Pharmacological Activity And Mechanism Of Ligustrazine Derivatives

Compound 17,4,13,26 is produced by Department of Pharmacy Beijing University of Chinese Medicine.This paper got the financial support from National Natural Science Foundation of China(No.81173519)and Innovation Team Project Foundation of Beijing University of Chinese Medicine named 'Lead Compounds Discovering and Developing Innovation Team Project Foundation'(No.2011-CXTD-15).The experiment was divided into two parts.Part one Pharmacological activity and mechanisms of compound 17 on ischemic strokeCompound 17 is a TMP structural analog,which produced with TMP active ingredient according to the composition principle,based on the theory of Chinese traditional medicine.This compound was selected from 60 kinds of TMP derivatives,with a strong activity on ischemic stroke.the subject intends to establish an vitro model of ischemic damage nerve cells by inducing differentiation of PC 12 cells,,in order to further explore the role of the drug and its mechanism.?.Effect of compound 17 in PC 12 cells and its mechanismPurpose:Effects of compound 17 was observed on differentiated PC 12 cells which induced by NGF growing under the influence of ischemic injury and observe the expression of NF-KB/p65 and COX-2 in cells in order to explore the role of compound 17 on nerve cells and its mechanism.Method:1.Influences of Compound 17 on the activity of nerve cells:undifferentiated PC 12 cell growth conditions is 80%RPMI1640+5%horse serum+10%fetal calf serum+100?g·mL-1 streptomycin and 100U·mL-1 penicillin;differentiation medium is 90%RPMI1640+10%FBS+100?g·mL-1 streptomycin+100 U·mL-1 penicillin;cells growing at saturated humidity incubator with 37? and 5%CO2.Differentiated PC12 cells are induced by NGF and used to establish ischemic injury model with CoCl2 for 12 hours.The effect of compound 17 was observed by the models and the results were evaluated by MTT assay.2.Influences of compound 17 on ischemic neuronal apoptosis:The effect of different concention of compound 17 on differentiated PC 12 cells apoptosis with ischemic injury was estimated by flow cytometry.It was explored that whether different concentrations of compound 17 lead nerve cells apoptosis by promoting nerve cells proliferation in order to reduce the degree of damage of nerve cells.3.The effect of the different concentrations of Compound 17 on the morphology of differentiated PC 12 cells with ischemic injury and its mechanism study.:HE staining was used to observe the morphology changes;ICC staining was used to analysis the expression of NF-?B/p65 and COX-2.Results:1.PC12 cells culture system is proficiently mastered,the experimental results are stable;and we verify the feasibility of differentiated PC 12 cells models with ischemic injury which has been established;50ng·mL-1 NGF was administered to induce PC 12 differentiation to establish nervous cells model;200?mol·L-1 cobalt chloride was administered to differentiated PC 12 cells for 12h to establish nervous cells models with ischemic injury.2.The compound 17 can promote the activity of differentiated PC 12 cell with ischemic injury,the concentration of 60?mol·L-1 reached a peak,the OD value of concentration groups(3.75?mol·L-1,7.5?mol·L-1,15?mol·L-1,30?mol·L-1)was significantly higher than the model group and TMP group,the OD value of 60?mol·L-1 of significantly higher than the NGF group;3.Compound 17 can promote apoptosis of undifferentiated PC 12 with ischemic injury;different concentrations of compound 17 groups can significantly inhibit neuronal apoptosis,increased cells survival;cells survival rate of concentration groups(15?mol·L-1,30?mol·L-1,60?mol·L-1)was significantly higher than the NGF group and the model group.4.The impact of compound 17 on morphology of nerve cells with ischemic injury.HE staining results:(1)NGF group:Compared with model group,it showed that cell volume became bigger,the nucleus also became larger,almost every cells growing with short neurites of varying lengths and formed a network;(2)the model group:cell poles protruding decreased or disappeared;poor cell refractive poor,nuclear condensation,nuclear fragmentation,nuclear fusion,less cytoplasm,cell membrane rupture and cell necrosis;(3)compound 17 group:compared with the NGF group,it can be found that the length of cells neurites was prolonged,especially high-dose group changed significantly.ICC staining results:(1)NF-?B/p65:Compound 17 decreased the expression of NF-?B/p65 during hypoxia in nerve cells;(2)COX-2:Compound 17 reduced the expression of COX-2 during hypoxia in nerve cells.Discussion:Compound 17 may have anti-tumor activity and may have a specific protective effect against nerve cells with ischemia injury;compound 17 has NGF-dependent neurite outgrowth promoting activity;its mechanism may be as follows:Compound 17 may reduce the inflammatory response by decreasing NF-?B/p65 and COX-2 expression intended to protect PC12 cells with ischemia injury.?.ConclusionIn summary,compound 17 has obvious NGF agonist activity,and can significantly reduce damage after ischemia and hypoxia in differentiated of PC 12 cells with ischemic neuronal injury.As concentrations increasing,the protective of compound 17 significantly enhanced.Its mechanism may be as follows:promoting neuronal proliferation,inhibiting neuronal apoptosis,reducing expression of COX-2,thereby inhibiting inflammatory response after ischemic injury,resulting in neuronal protection.Part two Pharmacological activity and mechanisms of compounds 4,13,26 on tumor cells activitySelection of anti-tumor active ingredients from natural compounds,according to the combination principle,the synthesis of lead compounds,to improve their physical and chemical properties intends to significantly increased the antitumor effect and reduced toxicity,has become one of the ideas of the development of new anticancer drugs.TMP has pharmacological and physiological effects such as blood vessels dilation,platelet aggregation inhibition,coagulation,promote microcirculation,antioxidant and calcium antagonists,anti-vascular endothelial endothelin and protection and so on.Compound 4,13,26 derive from TMP which synthesized by Beijing University of Traditional Chinese Medicine College based on the composition principle.These compounds were selected from 60 kinds of TMP derivatives,with a strong anti-tumor activity.Acute toxicity test of the three kinds of compounds showed that these compounds were safe and had mild toxicity or even not appeared.MTT assay was used to assess the cytotoxicity of different concentrations of compounds 4,13,26 against HepG2,MCF-7,Hela and HT-29 tumor cells,and calculate the IC50,compared with positive drug DDP.AnnexinV-PI apoptosis detection kit and PI with flow cytometry were used to evaluate the effects of compound 4,13,26 on HepG2 cells apoptosis;and detecting the impact of compound 26 on HepG2 cell cycle distribution;H22 tumor-bearing mice models were used to investigate compound 26 on tumor growth inhibition,to provide experimental evidence for the discovery of anti-tumor lead compounds.1.Screening of Antitumor lead compounds and their IC50 values.In order to investigate lead compounds which we are studying,focusing on the anti-tumor activity of TMP derivatives,four kinds of tumor cells and MTT screening assess were used to evaluate the cytotoxicity of compounds 4,13,26 were further screened,and the IC50 values was also calculated.The results showed that the anti-tumor effects of TMP structure derivatives 4,13 and 26 were significant,they inhibited the proliferation of tumor cells and showed dose-dependent,and the IC50 values were almost the same with DDP.2.The compound 4,13 and 26 induced HepG2 cell apoptosis detectionIn order to study whether compounds 4,13 and 26 inhibit HepG2 cell proliferation by inducing cell apoptosis,we used flow cytometry to detected the change of cell apoptosis rate.The results showed that the compounds 4,13,26 can induce apoptosis in a concentration-dependent manner,and dominated the early apoptosis.3.Compound 26 influenced HepG2 cell cycle distribution detectionMost anti-tumor compounds induce tumor cells apoptosis both with cell cycle-dependent,so we used flow cytometry to detect the effect of compound 26 on HepG2 cell cycle distribution.The results showed that compound 26 can influences each phase of the cell cycle,cell percentage in each phase changed significantly,mainly for three different concentrations groups increased in GO/GI of G2/M phase,and the number of cells in S phase reduced.These results suggested that compound 26 allows cells arresting in GO/GI of the G2/M phase,stoped the cell in G2/M phase to migrate to S phase,ultimately induced cells apoptosis mainly in S phase.4.The antitumor effect of compound 26 on H22 tumor-bearing miceCompound 26 showed significant anti-tumor effect on inhibiting tumor cells proliferation by inducing apoptosis and disturbancing tumor cell cycle which utimately caused tumor cell death.To investigate the anti-tumor effect of compound 26 in vivo,H22 tumor-bearing mice models were used to evaluate the inhibition on tumor growth.The results showed that the compound 26 significantly inhibited tumor growth in tumor-bearing mice,the inhibition rate was significantly increased.It consisted to this results in the second part of the experiment and the second experiment which showed that the high concentration of compound 26(60?mol·L-1)can significantly increase the rate of apoptosis of tumor cells.It suggested that TMP structural derivatives 26 may induce cell apoptosis and inhibit tumor cell growth,its mechanism needed to be further explored.