| Objective: To study the inhibitory effect of Total Flavonoids of Hedyotis diffusa on tumor growth and immune related indexes in MFC gastric tumor-bearing mice,and to explore the potential mechanism of Total Flavonoids of Hedyotis diffusa in inhibiting gastric cancer,so as to provide theoretical reference for clinical application of Total Flavonoids of Hedyotis diffusa in preventing and treating tumor.Methods: Fifty-two male C57BL/6 mice,SPF grade,were inoculated with MFC cell suspension after disinfection in the right armpit to establish MFC gastric cancer bearing mice model.On the 6th day after inoculation,when the swollen node grows to about 5mm×5mm,two mice were randomly selected,and when all the mice were pathologically diagnosed as tumor tissues after dissection,the model was successfully established.Fifty tumor-bearing mice were randomly divided into model group,positive group,high,medium and low dose groups of Total Flavonoids of Hedyotis diffusa,with 10 mice in each group and 10 mice as blank group.Drug intervention was given from the 7th day after inoculation.The blank group was given volume of normal saline by gavage as same as the model group,and intraperitoneal injection,the positive group was given cyclophosphamide 25 mg·kg-1by intraperitoneal injection,and the high,medium and low dose groups were given Total Flavonoids of Hedyotis diffusa by gavage of 200 mg·kg-1,100 mg·kg-1and 50 mg·kg-1respectively,once a day for 8consecutive days.During the administration period,the general state of mice in each group was scored;On the 7th,8th,9th,11 th,13th and 15 th days after inoculation,the long and short diameters of mice in each group were measured with vernier caliper,and the tumor growth curve was drawn.After the last administration,the tumor and organs were completely removed,and the tumor inhibition rate and spleen and thymus index of mice in each group were calculated Histopathological changes of tumor were observed by HE staining.Serum CEA,CA72-4,IL-2,IL-6 levels were detected by ELISA.The expressions of TLR4,MyD88 and NF-κB in tumor tissues of mice in each group were detected by immunohistochemistry,and semi-quantitative analysis was carried out.Results:1.From the modeling method,this method has the advantages of safety,reliability,easy operation,convenient observation,short modeling time,etc.The tumor formation rate is 100%,which accords with the characteristics of experimental model and can meet the requirements of this experiment.2.From the general state score of mice,compared with the model group,the physical condition of mice in positive group decreased significantly,while that in high-dose group increased significantly（P<0.01）.There was no significant difference in body mass between the middle and low-dose groups（P>0.05）.Compared with the blank group,the comprehensive score of the model group mice decreased significantly（P< 0.01）.Compared with the model group,it was increased in each group（P<0.01 or P<0.05）.After administration,except for the positive group,mice showed depilation,and their mental state,activity and appetite were improved.3.From the tumor growth curve,compared with the model group,the tumor volume of the positive group and the high-dose group of Total Flavonoids of Hedyotis diffusa decreased significantly（P<0.05）,while the tumor growth of the middle and low-dose groups of Total Flavonoids of Hedyotis diffusa decreased,but the difference was not statistically significant（P>0.05）.4.From the observation of tumor body,tumor weight and tumor inhibition rate of mice,the tumor body of mice is spherical or irregular,which is a substantial mass.In the model group,the tumor volume is too large,and some of it adheres to the surrounding skin tissue,with hard texture and obvious blood vessels;The tumor volume of mice in the administration group gradually decreased,the boundary was clear,it was easy to peel off,and it was fish-like Compared with the model group,the tumor weight of each administration group was significantly reduced（P<0.01）,and the tumor inhibition rate of positive group was 73.40%,that of high-dose Hedyotis diffusa 66.60%,medium-dose group 60.09%,and low-dose group47.36%.5.From the pathological changes,the tumor cells in the model group are closely arranged,with stable morphology and structure,less nuclear shrinkage and no obvious necrosis and fragmentation;In the positive group,the tumor cells were loosely arranged,with different degrees of flaky necrotic areas,a large number of nuclear pyknosis,nuclear fragmentation and vacuoles.The tumor cells in each dose group showed nuclear pyknosis,cell necrosis and nuclear fragmentation in a dose-dependent manner.6.From the serum biochemical indicators,the levels of CEA,CA72-4 and IL-6 in the model group were significantly increased（P<0.01）,while the levels of IL-2 were significantly decreased.Compared with the model group,the levels of CEA and CA72-4 in serum of mice in positive group,high and medium dose groups of Total Flavonoids of Hedyotis diffusa decreased significantly（P<0.01）,while the low dose group of Total Flavonoids of Hedyotis diffusa showed a downward trend,but the difference was not statistically significant（P> 0.05）.Compared with the model group,IL-6 decreased and IL-2 content increased in the high and middle dose groups,while IL-2 and IL-6 decreased in the positive group（P<0.01 or P<0.05）.7.Compared with the model group,the expressions of TLR4 and MyD88 in the positive group,the high and middle dose groups of Total Flavonoids of Hedyotis diffusa were significantly decreased（P<0.01）,but there was no significant difference in the low dose group of Total Flavonoids of Hedyotis diffusa.The expression of NF-κB in positive group and high-dose group of Total Flavonoids of Hedyotis diffusa decreased significantly（P<0.01）.Conclusion:1.Total flavonoids of Hedyotis diffusa can inhibit the growth of tumor and reduce the level of tumor markers,which has a good anti-tumor effect.2.Total flavonoids of Hedyotis diffusa can regulate immune-related cytokines,maintain Th1/Th2 balance and improve immune function.At the same time,it can down-regulate the expression of TLR4,MyD88 and NF-κB in tumor tissues,suggesting that it may reverse tumor immune escape. |
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