Effect Of Prunella Vulgaris Extract On B Cell Lymphoma In Vitro And In Vivo And Its Mechanism

BackgroundLymphoma is one of the earliest discovered hematologic malignancies,which originated from lymph nodes and lymphoid tissues.Most of the lymphomas are related to the proliferation and differentiation of some immune cells in the immune response and is a kind of malignant tumor of the immune system.In recent years,the incidence of lymphoma in China is on the rise,and has been ranked among the top ten causes of death from malignant tumors.In China,the incidence of non-Hodgkin's lymphoma?NHL?is much higher than that of Hodgkin's lymphoma?HL?.Diffuse large B-cell lymphoma?DLBCLs?is the most common type of lymphoma.It can be divided into GCB DLBCLs,ABC DLBCLs and primary mediastinal DLBCLs,which are produced in different differentiation stages of B cells.Three different subtypes of DLBCLs have different prognosis.Combination chemotherapy is the main treatment method at present,which plays an important role in prolonging the survival time and improving the curative effect.Anti-CD20 monoclonal antibody Rituximab combined with cyclophosphamide,doxorubicin,vincristine and prednisone?R-CHOP?has achieved certain efficacy in DLBCL.Nevertheless,about 1/3 of DLBCL patients have not achieved lasting remission.In order to improve the effect of DLBCL,it is imperative to develop new treatment drugs.Prunella vulgaris is a kind of traditional Chinese medicine,which is widely used in reducing sugar,lowering blood pressure,antioxidation,antimicrobial,anti-inflammatory,anti-virus and anti-tumor etc.Because of its more pharmacological activities,the research on Prunella vulgaris is also more in-depth and extensive,especially its anti-tumor effect.Prunella vulgaris can be used to treat thyroid cancer,breast cancer,lung cancer,liver cancer,lymphoma and other tumours.The effects of Prunella vulgaris on malignant tumors include?1?improving clinical symptoms and quality of life,improving survival rate;?2?The effect of reducing toxicity,increasing sensitivity and increasing efficiency of chemotherapy and radiotherapy;?3?To improve the effect of surgical treatment and reduce the complications and sequelae of surgery;?4?Prevention of tumor recurrence and metastasis and prevention of precancerous lesions;?5?Antioxidant and immunomodulatory function;?6?Inhibition of tumor cell growth and induction of cell apoptosis.The research on Prunella vulgaris has been widely studied,but as the highest incidence of DLBCLs,there are no reports on the effect of Prunella vulgaris in vivo and in vitro.ObjectiveTo study the biological function of Prunella vulgaris extract on DLBCL cell line ly8 and its mechanism.Materials and methods1.Cell culture:the cell line was cultured on a complete medium and placed in a incubator containing 5%CO₂ at 37?with relative humidity of 95?.The medium is changed or added every 2-3 days according to the condition of the cells.2.Cell proliferation:CCK8 assay was used to detect the cell proliferation.Different concentrations of Prunella vulgaris extract,the positive control drug vincristine?VCR?and the solvent control acted on the cells for 48 hours,then added the CCK8 detection solution to determine the absorbance of the wavelength in 450nm by enzyme standard instrument.3.Apoptosis:Apoptosis was detected by Annexin V/7AAD assay after 48 hours of treatment with the extract of Prunella vulgaris and VCR.4.Detection of PI3K/AKT/m TOR pathway related proteins and apoptosis related proteins by Western blot:The expression of related proteins was detected after 48hours of drug treatment on ly8 cell lines.5.Ly8 tumor model was established in mice.The mice were randomly divided into 5 groups with 6 mice in each group:The Prunella vulgaris extract in the high,medium and low dose groups?400 mg/kg/d?200 mg/kg/d?100 mg/kg/d?were injected intraperitoneally respectively for 8 days;Positive control group?intraperitoneal injection of vincristine 0.5 mg/kg/d,day 1?;Negative control group?intraperitoneal injection of isobaric solvent,continuous injection for 8 days?.To observe the inhibition rate of Prunella vulgaris extract on tumor-bearing mice.6.Statistical analysis:the experimental data were analyzed by SPSS 21.0software.The measured data were all expressed as mean±standard deviation??x±s?.Single factor variance analysis was used to analyze the differences between groups,p<0.05.The software GraphPad Prism 6.0 was used to plot.Results:1.The extracts of Prunella vulgaris had inhibitory effects on the proliferation of three B-cell lymphoma cell lines,all of which had statistical significance compared with the control group?P<0.05?.With the increase of the extract concentration of Prunella vulgaris,the inhibition rate was higher and the inhibitory effect was stronger,which indicated that the drug had dose-effect dependence in a certain range of drug concentration.The inhibition rate of the drug on the three cell lines can reach more than 95%.The average IC₅₀0 of the three cell lines was:LY8 20.9?g/ml,RAJI 29.89?g/ml,DOHH2 38.89?g/ml,LY8 cells were the most sensitive to the Prunella vulgaris extract with the lowest IC₅₀0 value.2.The effect of Prunella vulgaris extract on apoptosis of LY8 cell line was observed.Compared with solvent control group,the apoptotic rate of LY8 cells in Prunella subtilis extract group was significantly higher than that in control group.3.Effect of Prunella vulgaris extract on PI3K/AKT/mTOR pathway:After treated with Prunella vulgaris extract for 48 hours,Western-Blot analysis was performed.Compared with the blank control group,the expression of total AKT protein in Prunella vulgaris extract group was not decreased,but the expression of phosphorylated AKT protein was decreased,and the expression of m TOR protein in its downstream protein was also decreased.The results showed that the Prunella vulgaris extract could inhibit the PI3K/AKT/mTOR pathway.4.Effect of Prunella vulgaris extract on apoptosis related protein:After 48 hours of treatment with Prunella vulgaris extract on LY8 cells,Western-Blot analysis was carried out.Compared with the blank control group without drugs,the expression of apoptotic protein Cleaved caspase-3 were increased in the Prunella vulgaris extract group.5.In vivo experiment:At the end of the experiment,the volume of tumor in solvent control group was 1556.03±181.81mm³,The tumor volume of high dose group,middle dose group and low dose group were 626.79±85.43mm³,957.45±107.54mm³,1387.65±110.81mm³ respectively;The inhibition rates of tumor growth were 70.33%,45.23%,12.59%respectively.Compared with the solvent control group,the difference between the high dose group and the middle dose group was statistically significant?P<0.05?,but there was no significant difference between the low dose group and the low dose group?P>0.05?.Conclusions:1.The extract of Prunella vulgaris inhibited the PI3K/AKT/mTOR pathway and activated the apoptosis-related protein to exert anti-tumor effect;2.The extract of Prunella vulgaris has obvious antitumor effect on ly8tumor-bearing mice and has mild toxicity.