| Research PurposeBased on our preliminary clinical application of the Modified Shengma Biejia Decoction(SMBJD)in the adjuvant treatment of patients with multiple myeloma(MM),we found that it could obviously improve the therapeutic effect.However,itsspecific molecular biological mechanism hasn’t been clarified.In this study,two human multiple myeloma cell lines,H929 and U266,are used to explore the effect of SMBJD on the proliferation of the two cell lines in vivo and in vitro.On this basis,a series of experiments are used to explore the molecular mechanism of its biological effect,so as to elaborate the scientific basis of SMBJD in the treatment of MM.Research Methods Methods of in vivo study:1.H929 cells were collected and adjusted it’s density to a concentration of 5×107 cells/mL.Each mouse in the experimental group was injected with 0.2 mL(1×107 cells)into the left forelimb to establish H929 xenograft mouse model.When the tumor volume of most nude mice reached 80-100 mm3,the standard mice were randomly divided into the model group,low-dosage SMBJD treatment group and high-dosage SMBJD treatment group.They were given intragastric administration of sterile double distilled water(0.2 mL/d,once per day),low-dosage SMBJD(3.03 g/mL,0.2 mL/d,once per day)and high-dosage SMBJD(6.07 g/mL,0.2 mL/d,once per day)for 7 days respectively.Observed the tumor growth of each groups;2.Measured the length and width of tumors every 3 days,then calculated the tumor volumes and observed the mental states of each mice at the same time;3.24 hours after the last gavage,collected the blood of each mice through the retro-orbital sinus and detected after anesthetized them.At last,sacrificed them by cervical dislocation;4.HE staining was used to detect the histological changes of liver,spleen and kidney of each mice;5.Western blot(WB)was used to detect the expression of P62,LC3,Bax,Bcl-2,caspase-3 and cleaved Caspase-3,as well as the ERK/mTOR signaling pathway related proteins;6.Immunohistochemistry(IHC)was used to detect the expression of LC3 and Cleaved Caspase-3 in tumor tissues.Methods of in vitro study:1.The main active components of SMBJD were detected through High performance liquid chromatography(HPLC);2.The viability of H929 and U266 after the treatment of different concentrations of SMBJD(1,2,4,8,16 mg/ml)for 36 h and 48 h respectively were analysis through MTT assay.After that,calculated and compared the IC50 value of SMBJD on the two cell lines at different intervention time;3.Ad-GFP-LC3 was transfected to labeled LC3 protein.After 36 hours intervention with different final concentrations of SMBJD(1,2,4 mg/ml),counted the number of autophagosomes of each cell under fluorescence microscope;4.The effect of SMBJD on the apoptosis of H929 and U266 cells was analysis by Annexin V-FITC/PI double staining flow cytometry(FCM);5.The expression of P62 and LC3,Bax,Bcl-2,Caspase-3,cleaved caspase-3 and ERK/mTOR signaling pathway related proteins in H929 and U266 cells after 36 hours of intervention with SMBJD(1,2,4 mg/ml),3-MA(0.25 mmol/L)and Baf-A1(0.5 nmol/L)were detected through WB assay;6.After SMBJD(2 mg/ml)alone or combined with Rapamycin(1 nmol/L),a specific inhibitor of mTOR,was intervened H929 and U266 cells for 36 h,and then the expression of autophagy-and apoptosis-related proteins were detected by WB assay.Research Results Results of in vivo study:1.According to the measurement results,the curves of body weight and tumor volume were drawn.After 7 days of administration,the mice in the model group were lighter compared with the blank group(P<0.05).While the mice in the two SMBJD treatment groups were heavier compared with the model group(P<0.05).As for the tumor volume,the results showed that SMBJD could significantly inhibit the tumor growth compared with the model group(P<0.05).2.The results of blood routine analysis of nude mice in each groups showed that WBC and PLT of the model group were significantly increased(P<0.05),while the RBC and Hb significantly decreased(P<0.05)compared with the blank group.Moreover,the WBC,RBC and Hb in the SMBJD treatment groups were significantly improved(P<0.05)except for PLT compared with the model group.While compared with the blank group,there was no significant difference of WBC,RBC and Hb(P>0.05)except for PLT in the SMBJD treatment groups.3.The results of HE staining showed that the liver,spleen and kidney in the model group had obvious pathological damage,while the damage of organs in the SMBJD treatment groups was significantly reduced.4.The results of WB assay indicated the expression of P62 increased(P<0.05),the ratio of LC3-II/LC3-I decreased(P<0.05),while the ratio of Bax to Bcl-2 and Cleaved Caspase-3 to Caspase-3 increased gradually(P<0.05).In addition,the expressions of ERK/mTOR signaling pathway related proteins p-ERK and p-mTOR also significantly increased in low-dosage and high-dosage SMBJD groups(P<0.05)compared with the model group.5.The results of Immunohistochemistry(IHC)showed that the expression of autophagy key protein LC3 in the two SMBJD treatment groups decreased(P<0.05),while the expression of Cleaved Caspase-3 increased(P<0.05)compared with the model group.Results of in vitro study:1.The five major components of SMBJD(1.0 g/mL)were analyzed by HPLC.The liquiritin,ferulic acid,cimifugin,isoferulic acid,and glycyrrhizic acid contents were 1102.20,229.24,67.14,1534.73,and 1560.82 μg/g,respectively.2.MTT assay showed that SMBJD(1,2,4,8,16 mg/mL)could inhibit the growth of H929 and U266 cells in a dose-and time-dependent manner.The IC50 value of SMBJD to H929 and U266 cells were 6.16±0.14 mg/ml and 6.02±0.29 mg/ml for 36 h,3.44±0.79 mg/ml and 3.83±0.33 mg/ml for 48 h,respectively.However,there was no significant difference in IC50 values between the two cell lines at the same intervention time(P>0.05).3.Fluorescence-labeled autophagosomes were counted after infection with Ad-GFP-LC3 to detect levels of autophagy.There was a substantial accumulation of autophagosomes in both H929 and U266 cells.However,numbers of GFP-LC3 puncta were decreased after treatment with SMBJD compared with the control group(P<0.05).4.The Flow cytometry(FCM)results showed that SMBJD ould induce H929 and U266 cells apoptosis in a concentration dependent manner(P<0.05).5.After treated with different concentrations of SMBJD(1,2,4 mg/mL)for 36 h,WB assay was used to analysis the expression of autophagy-and apoptosis-related proteins in each groups.The results showed that SMBJD could significantly increase the expression of P62,the ratio of Bax/Bcl-2 and Cleaved Caspase-3/Caspase-3,reduce the ratio of LC3-II/LC3-I with the increase of SMBJD concentration compared with the control group(P<0.05).Meanwhile,the expression of ERK/mTOR signaling pathway related proteins p-ERK and p-mTOR increased gradually with the increase of SMBJD concentration compared with the control group(P<0.05).6.The expression of LC3 in H929 and U266 cells treated with SMBJD(2 mg/ml)alone or in combination with 3-MA(an early-stage autophagic inhibitor)and Baf-A1(a late-stage autophagic inhibitor)for 36 h was detected by WB assay.The results showed that compared with the control group,both SMBJD and 3-MA could reduce the ratio of LC3-Ⅱ/LC3-Ⅰ in H929 and U266 cells(P<0.05),while the ratio of LC3-Ⅱ/LC3-Ⅰ in Baf-A1 group was significantly increased(P<0.05).In addition,there was no significant difference in LC3-Ⅱ/LC3-Ⅰ ratio between SMBJD+Baf-A1 group and Baf-Al group(P>0.05).7.After SMBJD(2 mg/ml)alone or combined with Rapamycin(a specific inhibitor of mTOR)intervened H929 and U266 cells for 36 h.The results of WB assay showed that compared with the control group,Rapamycin could reduce the expression of P62 and increase the ratio of LC3-Ⅱ/LC3-Ⅰ(P<0.05),but had no effect on the ratio of Bax/Bcl-2 and Cleaved Caspase-3/Caspase-3(P>0.05).Compared with SMBJD group,the expression of P62 was decreased and the ratio of LC3-Ⅱ/LC3-Ⅰ was increased significantly(P<0.05),while the ratio of Bax/Bcl-2 and Cleaved Caspase-3/Caspase-3 significantly decreased in SMBJD+Rapamycin group(P<0.05).Research Conclusion1.The five major components(liquiritin,ferulic acid,cimifugin,isoferulic acid,and glycyrrhizic acid)of SMBJD(1.0 g/mL)were analyzed by HPLC.2.The anti-tumor effect of SMBJD in vivo and in vitro may be achieved by inhibiting the protective autophagy and inducing autophagy-mediated apoptosis of H929 and U266 cells.ERK/mTOR signaling pathway may be involved in the regulation of this process as an upstream regulatory signal. |
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