| Objective:The bone marrow microenvironment of leukemia host is beneficial to the proliferation of leukemia cells,but not to the normal hematopoiesis.The inflammatory transition is an important reason,some leukemia host bone marrow inflammatory factors and(Wip1)genes are significantly increased.Previous studies have confirmed that Compound Fritillaria Scutellaria Decoction(CFSD)can reduce Wip1,inhibit leukemia cell proliferation and improve inflammatory response,suggesting that the bone marrow inflammatory microenvironment is involved in the process of CFSD to inhibit proliferation.Taking patients with B-cell lymphoma as the retrospective clinical research object,the relationship between serum IL-6 and lymphoma efficacy is preliminarily discussed.This subject takes the L1210 lymphocytic leukemia mouse model as the research object,through cell ’biology and molecular biology techniques,to further confirm that the expression of bone marrow inflammatory factors in leukemia state is significantly increased.Taking CFSD as an intervention measure,the project detects the leukemic remission,IL-6,TNF-α,and Wip1mRNA in mice to explain the mechanism of CFSD in improving bone marrow microenvironment by promoting the elimination of inflammatory factors.By clarifying the influence of CFSD on bone inflammatory marrow microenvironment,we try to seek new targets for the treatment of leukemia and lymphoma with traditional Chinese medicine.Method:1.Retrospective clinical study:To explore the relationship between IL-6 and the curative effect of lymphoma,and to explore the advantageous group of CFSD,non-Hodgkin’s lymphoma patients in the Department of Hematology and oncology of Dongzhimen Hospital of Beijing University of Chinese medicine were selected as the research objects From January 2013 to December 2018.Inclusion criteria:①diagnosed as non Hodgkin’s lymphoma from B cell;②treatment plan:CFSD combined with 2-cycle standard chemotherapy;③serum IL-6 was detected before and after 2-cycle chemotherapy;④No infectious diseases were detected within one week before the detection of serum IL-6;⑤therapeutic effect was evaluated after 2-cycle treatment.To analyze whether the change trend of serum inflammatory factor IL-6 can predict the curative effect of lymphoma.The collected cases were used to analyze whether the change trend of serum IL-6 could predict the curative effect of lymphoma.2.Model:Acute lymphoblastic leukemia model was established by tail vein inoculation.After breeding 5-6 week-old BALB/c mice for 1-2 days to acclimate to the environment.L1210 cell suspension was extracted(1×107 per mouse),and inoculated via the tail vein of mice to form a systemic disseminated leukemia model.The general condition of the mice was observed.Peripheral blood was collected through the tail vein on days 0 and 10 for whole blood cell analysis,peripheral blood leukocyte classification,and blood smears were prepared to identify the mouse-model.3.Group administration:BALB/c mice were randomly divided into three groups,six mice in each group:TCM group,model group and normal group.After L1210 mouse model was established successfully,the traditional Chinese medicine group was given CFSD by gavage.According to the conversion coefficient of mice,coefficient was 8.65 times.After the conversion,the concentration of traditional Chinese medicine was 13.30ml/kg,i.e.0.20ml/d,and it was administered continuously for 15 days.The normal group and the model group were given equal volume distilled water once a day for 15 days.4.Observation index:Observe the general condition and survival status of mice in each group.Peripheral blood was collected before group administration and on day 1,8,15 or after dying.On the 15th day or before dying,blood was collected from the orbit to detect blood routine and prepare peripheral blood smear.IL-6 and TNF-α in bone marrow and peripheral blood were detected by ELISA,WiplmRNA in bone marrow and peripheral blood were detected by RT-qPCR,and pathological sections of liver,spleen and kidney were stained with HE to observe the structure and cell morphology.Influence of inflammatory factors and histopathological changes in leukemia mice were explored.Result:1.The serum IL-6 of lymphoma patients increased after CFSD combined with two-cycle chemotherapy,and the serum IL-6 in the progression group group was significantly higher than before treatment(P<0.05).Too high serum inflammatory factor IL-6 may indicate an adverse outcome of lymphoma,which can be further confirmed by a large sample study.①A total of 14 patients with B cell-derived non-Hodgkinlymphoma(NHL)were fit,including 7 males and 7 females with a median age of 65 years.There were 9 cases of invasive lymphoma,5 cases of non-invasive lymphoma;4 cases of Ann-Arbor stage ≤Ⅱ,and 10 cases of stage≥Ⅲ.IPI score was<3 in 3 groups and IPI index was>3 in 11 patients.②According to gender,median age,invasiveness,Ann-Arbor staging,and IPI scores,there were no significant differences in serum IL-6 concentrations(P>0.05).③After treatment,serum IL-6 increased significantly:the serum IL-6 concentration was(8.50±6.90)pg/ml before treatment and(24.66±27.28)pg/ml after treatment,which was significantly higher than that before treatment(P<0.05).④Comparing the trend of IL-6 according to clinical efficacy.Divided into treatment benefit group:4 cases in remission group and 3 cases in stable group;disease progression group:7 cases.The concentration of IL-6 was(7.58±4.06)pg/L and(9.41±8.79)pg/L before treatment in the benefit group and the disease progression group respectively.the baseline levels were the same.The concentration of IL-6 in the peripheral blood was(25.54±31.38)pg/L and(26.77±22.24)pg/L after treatment respectively.There was no significant difference in IL-6 concentration between the two groups before and after treatment,but the serum IL-6 concentration in the progressive group was significantly higher than before treatment(P<0.05).In the treatment-benefiting group,the pre-treatment serum IL-6 concentrations in the remission group and the stabilization group were(8.10±3.19)pg/ml,(6.89±4.91)pg/ml,respectively.And there were(14.39±15.36)pg/ml,(33.41 ±42.16)pg/ml after treatment.There was no significant difference in serum IL-6 concentration before and after treatment.Serum IL-6 in the disease progression group was significantly higher than that before treatment,which may indicate an unfavorable outcome of lymphoma,which needs to be further confirmed by a large sample study.2.L1210 leukemic mice model was established successfully.Eighteen SPF male BALB/c mice were randomly divided into three groups,six in each group:model group,traditional Chinese medicine group and normal group.Model group and traditional Chinese medicine group mice were inoculated with 1×107 L1210 cells in logarithmic growth period via tail vein,and normal group mice were injected with equal volume of normal saline via tail vein.On the tenth day after modeling,blood was taken through the tail vein to make a blood smear.In the model group and the traditional Chinese medicine group,the peripheral blood smear of mice showed proto lymphocyte,the proportion of peripheral blood leukemic cells in the model group was(10.67±1.57)%,and that in the traditional Chinese medicine group was(10.83± 1.91)%,there was no significant difference between the two groups(P>0.05).There was no abnormal lymphocyte in peripheral blood of normal group.3.CFSD significantly improved the survival status of L1210 leukemia mice,reduced leukemia burden,significantly lessened tumor infiltration,and could prevent and cure tumor anemia.① Survival status:The two groups of mice receiving the modeling showed a sluggishness,depression,a dull coat,wasting,and their four claws were not warm.Individual mice in the model group could reach induration in the lower abdomen and tail,and incontinence.Compared with the model group,the mice in the TCM group who received the intervention of CFSD had improved in diet,spirit and activity.Compared with the model group.The death time of the Chinese medicine group was later than that of the model group.(2)Weight changes:The mice in each group were weighed on the 15th day of intervention.The weights of the normal group,model group,and TCM group were-respectively(25.72±0.84)g,(23.42±1.21)g,and(23.95±1.07)g.The weight of mice in each group gradually increased with time.The weight gain of the TCM group was better than that of the model group.③Whole blood cell analysis:The white blood cell counts in the model group,the traditional Chinese medicine group and the normal group were(9.3 6±1.75)×109/L,(9.11 ±0.70)× 109/L and(6.92±0.91)×109/L,respectively,The lymphocyte counts in the model group,TCM group and normal group were(6.3810.78)×109/L,(5.77±1.49)×109/L,(2.55±1.33)×109/L.The white blood cell and lymphocyte counts of the model group and the traditional Chinese medicine group were significantly higher than that of the normal group(P<0.05);while the white blood cell and lymphocyte counts of the traditional Chinese medicine group were lower than the model group,the difference was not statistically significant.The hemoglobin of model group,traditional Chinese medicine group and normal group were(140.25±31.20)g/L,(163.25±14.10)g/L,(162.83±4.91)g/L,respectively.Compared with the normal group,the hemoglobin in the traditional Chinese medicine group did not decrease,but that in the model group decreased by 20 g/L.④Pathology of spleen and liver:The spleen weight of the model group,TCM group,and normal group was(0.12±0.01)g,(0.09±0.01)g,(0.09±0.02)g,respectively.The spleens of the mice in the model group and the traditional Chinese medicine group all had different degrees of swelling,and tumor infiltration and necrosis could be seen.The spleen weight of the model group was the largesr than TCM group,P<0.05.In the traditional Chinese medicine group,the degree of spleen swelling was light,the tumor infiltration and necrosis were less than those in the model group.There were no significant differences in liver weight among the groups.In the model group and the traditional Chinese medicine group,the liver tissue structure was destroyed.In the model group,there were many ashen infiltrating and necrotic foci on the liver surface.In the traditional Chinese medicine group,the liver surface was rough,and the number and area of tumor infiltrating foci were less than that in the model group.The results showed that CFSD could reduce the load of leukemia,decrease the infiltration of tumor,and prevent and treat tumor anemia.4.The expression of IL-6,TNF-α and Wip1mRNA in the bone marrow microenvironment of L1210 leukemia mice was significantly increased.CFSD can significantly reduce the bone marrow and serum IL-6 and TNF-α,but did not reduce the expression of bone marrow Wip-mRNA.①Serum IL-6 and TNF-α:The expression of serum IL-6 in the model group,TCM group and normal group were(45.47±12.88)ng/L,(23.91 ±7.44)ng/L,(26.73±2.15)ng/L.Serum TNF-α were(65.79±19.77)ng/L,(36.65±15.88)ng/L,and(38.68±8.79)ng/L respectively.The expression of IL-6 and TNF-α in the serum of the traditional Chinese medicine group was significantly lower than that of the model group(P<0.05).The traditional Chinese medicine group was basically the same as the normal group.②IL-6 and TNP-α in bone marrow:The bone marrow IL-6 expression in the model group,Chinese medicine group,and normal group was(56.33±20.73)ng/L,(21.34±5.08)ng/L,and(27.40±3.59)ng/L.The bone marrow TNF-α expression was(40.66±14.47)ng/L,(24.17±2.94)ng/L,and(18.49±2.97)ng/L,The expressions of IL-6 and TNF-α in the bone marrow of the TCM group were significantly lower than those of the model group(P<0.01).③Explore the correlation between inflammatory factors and Wip1mRNA:The expression of Wip1 mRNA in bone marrow of leukemia mice was significantly increased.The relative expression of bone marrow Wip1mRNA in the model group,traditional Chinese medicine group and normal group were(5.88±6.32),(3.96±1.95),(1.00±0.56),respectively.The expression of bone marrow Wip1mRNA in the model group and the traditional Chinese medicine group was higher than that in the normal group,P<0.05.There was no significant difference in the expression of bone marrow Wip1mRNA between the traditional Chinese medicine group and the model group,P>0.05.The expression levels of bone marrow Wipl and inflammatory factors did not show significant correlation.,P>0.05.Conclusion:Under the condition of leukemia,the expression of bone marrow inflammatory factors is significantly increased.CFSD can reduce tumor burden by improving the bone inflammatory marrow microenvironment in mice with lymphocytic leukemia,and help prevent and treat tumor-related anemia. |
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