Clinical Evaluation Of Qinhuang Mixture In The Treatment Of Non-Hodgkin Lymphoma And Its Preliminary Mechanism Study

Background The incidence of malignant lymphoma is increasing rapidly,lymphoma has become one of the most common ten malignant neoplasms.Currently the conventional methods of treatment have shortcomings such as immunosuppressive associated side effects and drug resistance.We need to find a safer,more effective and economical drug to prevent and treat malignant lymphoma to improve prognosis and quality of life.Previous study found that Qinhuang Mixture combined with conventional treatment can improve the primary high-risk DLBCL immediate effect,achieved earlier complete remission,reducing the side effects of chemotherapy.The main components of the baicalin mixture are the flavonoids such as wogonin.A large number of studies have shown that flavonoids can inhibit tumor cells by inducing apoptosis and autophagy.The purpose of this study was to evaluate the effect of Qinhuang Mixture on non Hodgkin’s lymphoma from the angle of immunomodulating and improving quality of life.In addition,we also investigated the inhibitory effect and mechanism of some flavonoids on lymphoma cells from the perspective of apoptosis and autophagy.Objective 1.Evaluate the effect of Qinhuang Mixture on quality of life and immune function in patients with non Hodgkin’s lymphoma.2.Investigate the inhibitory effects and mechanisms of wogonin,luteolin and silybin on lymphoma cells.Methods 1.Patients were volunteered into the group according to real-world study methods,the patients with non-Hodgkin’s lymphoma treated or followed up in our hospital from January 2015 to December 2016 were divided into observation group and control group.Patients in observation group received Qinhuang Mixture treatment during routine chemotherapy or follow-up period.While patients in the control group did not take Qinhuang Mixture during routine chemotherapy and follow-up period.The observation period was 6 months.All patients reached the end of the study,the Kanofsky score(KPS),quality of life score(EORTC-QLQ-C30),peripheral blood cell immunity,humoral immunity and immune-related cytokines were compared between two groups.2.CCK-8 detection was taken to detect the effect of wogonin,luteolin,silybin,kaempferide,quercetin,apigenin,genistein on the proliferation of lymphoma SUDHL-4 cells,the IC50 value of flavonoids with better inhibition effect were calculated.Occurrence of apoptosis was detected by flow cytometry combined with Annexin V/PI double staining method.Fluorescence probe DCFH-DA combined with flow cytometry detected reactive oxygen species generation.The antioxidant NAC was used to verify the killing effect of ROS.Western blot detected the protein expression levels of cleaved caspase3,cleaved PARP,Cytochrome c,Bax,Bcl-2,LC3 A,LC3B,Beclin1,SQSTM1/p62 and other proteins.Results 1.A total of 222 cases of effective cases were collected and 74 cases were observed in the observation group,of which 20 were in the chemotherapy period and 54 were in the follow-up period.There were 148 cases in the control group,of which 87 were in the chemotherapy period and 61 were in the follow-up period.After treatment,the improvement rate of KPS score in the observation group was significantly higher than that of the control group(P < 0.05).The scores of fatigue,constipation and diarrhea in the observation group were lower than those in the control group(P < 0.05)in the Quality of Life(EORTC-QLQ-C30)Score.The scores of body,emotional function and overall health were higher than those in the control group(P < 0.05).After treatment,CD3+ level,CD4+ level and the ratio of CD4+/CD8+ and immunoglobulin G in peripheral blood T lymphocyte subsets(Immunoglobulin G,Ig G),immunoglobulin M(Ig M),interleukin 2(Interleukin 2,IL-2)and complement C3 levels of the observation group were higher than the control group(P < 0.05),the levels of interleukin 6(IL-6),interleukin 8(IL-8)and interleukin 10(IL-10)were lower than those in the control group(P < 0.05).2.Wogonin,luteolin and silybin all inhibited the proliferation of SUDHL-4 cells in a dose and time dependent manner.25,50,100 μM of wogonin,luteolin can induce apoptosis of SUDHL-4 cell,wogonin group apoptosis rates were(8.43 + 0.57%),(17.90 + 1.40%),(35.07 + 1.72%),respectively.The apoptosis rates of luteolin were(15.93 + 0.82%),(23.67 + 1.49%),(24.20 + 1.82%),there was statistically significant difference compared with control group(P < 0.01).25,50 and 100 μM of silymarin did not induce apoptosis of SUDHL-4 cells.The apoptotic rate of silymarin group were(3.89 + 0.62%),(3.38 + 0.39%),(3.49 + 0.64%),which were not significantly different from those of the control group(P > 0.05).wogonin can induce the accumulation of ROS(reactive oxygen species),and luteolin did not induce the accumulation of ROS.Wogonin and luteolin treatments on SUDHL-4 cells could up-regulate the expressions of cleaved PARP,cleaved caspase3,cleaved caspase8,cleaved caspase9,Cytochrome c and Bax,and down-regulate the expression of Bcl-2 and the ratio of Bcl-2/Bax.Silybin can up regulate the expression of Beclin-1 and down regulate the expression of SQSTM1/p62 and the ratio of LC3A/LC3 B in SUDHL-4 cells.Conclusions 1.Qinhuang Mixture treatment can effectively improve the quality of life and immune function of patients with non Hodgkin’s lymphoma.2.Wogonin,luteolin and silybin all inhibited the proliferation of SUDHL-4 cells.Wogonin could induce ROS accumulation in SUDHL-4 cells.Wogonin and luteolin induced apoptosis in SUDHL-4 cells,and silybin induced autophagy in SUDHL-4 cells.