Study On The Anti-tumor And Chemotherapy-induced Immunosuppressive Effects Of Guilu Erxian Jiao Decoction On H22 Tumor-bearing Mice

Objective From the PD-1/PD-L1 signaling pathway,through experimental studies,a tumor-bearing mouse model was established using the H22 liver cancer cell line subculture and transplantation method to detect tumor growth rate and tumor-bearing mice in tumor-bearing mice after drug intervention.Apoptotic ratio of spleen T lymphocytes and positive rate of PD-1 expression in infiltrating cells around the tumor of tumor-bearing mice;TGF-?1-si RNA inhibits PD-1 signaling pathway as a control,and in vitro observation of Guilu Erxian Jiao Decoction on mouse T lymphocytes The effect of PD-1 m RNA and protein expression in cell line CTLL-2.To investigate the molecular mechanism of Guilu Erxian Jiao Decoction in regulating tumor cell immunity and analyze whether it has protective effect on cisplatin-induced T cell injury,and to provide scientific basis for the adjuvant treatment of malignant tumors by Chinese medicine.Methods2.1Building model and cell suspension: The H22 hepatoma cell line was subcultured and transplanted to construct a mouse model of transplanted tumor.The passaged H22 hepatoma cell line was routinely cultured and inoculated in the abdominal cavity of SPF grade BALB/c.After 6 days,the mice were sacrificed.Ascites was aseptically aspirated and diluted into tumor cells with PBS.Suspensions were collected by centrifugation,subcutaneously inoculated into the axilla to construct an animal model of subcutaneous xenografts in mice;CTLL-2 cells were treated with 10% fetal bovine serum,100 U/ml of IL-2,and 1% double antibody(Streptomyces lividans).The RMPI-1640 broth culture medium was incubated in a 37°C,5% CO2 incubator.Observe the cells under the microscope as suspension cells.2.2 grouping drugs:2.2.1 Grouping administered in vivo: The tumor mice were randomly divided into cisplatin group,the high dose group of Guilu Erxian Jiao Decoction,the middle dose group of Guilu Erxian Jiao Decoction,the low dose group,and the saline group,with 3 animals in each group:(1)cisplatin group(chemotherapy control group): intraperitoneal injection of cisplatin,each 3mg/kg,gavage normal saline,each 0.3ml;(2)the high dose group of the Guilu Erxian Jiao Decoction: intraperitoneal injection of physiological saline,each 0.2ml,and gavage of the tortoise deer dixix 0.38g;(3)the middle dose group in the Guilu Erxian Jiao Decoction: intraperitoneal injection of physiological saline,each 0.2ml,and the gastric turtle deer dixime gum 0.19g;(4)the low-dose group: intraperitoneal injection of saline,0.2ml per dose,and the gastrodia elensis gum 0.095g;(5)physiological saline group(negative control): intraperitoneal injection of physiological saline,each 0.2ml,gavage physiological saline,each 0.3ml;After 10 days,the drug was administered to the drug for 9 days.2.2.2 Grouping administered in vitro: H22 hepatoma cells were added into the 6 Hole cell culture plate.After 24 h,the CTLL-2 suspension was transfected into 5 * 106 CTLL-2 cells in each pore after transfection of TGF-?1-si RNA.Divided into Guilu Erxian Jiao Decoction group + TGF-?1-si RNA group,Guilu Erxian Jiao Decoction group,cisplatin + TGF-?1-si RNA group,cisplatin plus Guilu Erxian Jiao Decoction + TGF-?1-si RNA group,cisplatin plus Guilu Erxian Jiao Decoction group,cisplatin group,cell control + TGF-?1-si RNA group,the cell control way,each group of three media:(1)cisplatin + Guilu Erxian Jiao Decoction group: the cells were treated with the culture medium containing 10 u M cisplatin and 6% turtle deer,and the serum of 4% fetal bovine serum.(2)cisplatin + Guilu Erxian Jiao Decoction +TGF-?1-siRNA group: transfected TGF-?1-si RNA,and then treated the cells in the medium containing 10 u M cisplatin and 6% of the rat serum(4% fetal bovine serum)after 24h;(3)cisplatin group: the cell was treated with a medium containing 10 u M cisplatin;(4)cisplatin +TGF-?1-siRNA group: transfected TGF-?1-siRNA,and treated cells with 10 u M cisplatin after 24h;(5)the group of Guilu Erxian Jiao Decoction: cultured cells cultured in rat serum(4% fetal bovine serum)with 6% turtle deer;(6)TGF-?1-siRNA group: TGF-?1-si RNA was transfected,and the cells were treated with a culture medium containing 6% of the rat serum(4% fetal bovine serum)after 24 h.(7)cell control +TGF-?1-si RNA group: transfection TGF-?1-si RNA;(8)control group: only complete medium;37 ? incubator 6 hours.2.3 Testing indicators2.3.1 tumor retesting of tumor-bearing mice: the tumor size was measured with a vernier caliper in the 1,4,7,10,13,16 and 19 days after the administration of the drug.After 29 days of tumor,the nude mouse was executed and photographed.The tumor was weighed and recorded,and the spleen was preserved.2.3.2 tumor-burdened mice spleen T lymphocyte apoptosis ratio detection: first collect a tumor-burdened mice spleen T lymphocytes cells due to the detection of T lymphocyte apoptosis and tumor infiltrating T cells PD-1 + proportion;2.3.3 m RNA and protein expression detection of PD-1/PD-L1/PD-L2 in vitro: real-time PCR technology was used to detect the influence of the expression of PD-1 / PD-L1 m RNA around the cell after experimental drug intervention.Western Blot method was used to test the influence of the expression levels of PD-1/PD-L1 and other related proteins in vitro cultured malignant tumor cells after experimental drug intervention.3 Results3.1Fourteen days later,compared with the normal saline control group,the cisplatin group,the high dose group of Guilu Erxian Jiao Decoction,the dose group of the Guilu Erxian Jiao Decoction were obviously inhibited the growth of the tumor(P<0.05),and the inhibitory tumor effect of the high dose group was significantly higher than the low dose group of P<0.05).3.2 The early T lymphatic apoptosis ratio in the high,middle and low dose groups was significantly lower than that in the cisplatin group and the normal saline(negative control)group.The difference was statistically significant(P<0.05).The lower dose group in the Guilu Erxian Jiao Decoction and the high dose group had a more obvious effect on reducing the apoptosis of the early T lymphoblastic cells(P<0.05),but in the Guilu Erxian Jiao Decoction group,There was no significant difference in the effect between the high dose group and the high dose group(P<0.05),and the late apoptotic cells in the high dose group were significantly lower than that of the cisplatin group and the normal saline(P<0.05)group(P<0.05).3.3 cisplatin group,Guilu Erxian Jiao Decoction in high dose group,the Guilu Erxian Jiao Decoction dosage group of PD-1 express positive cells than significantly lower compared with normal saline group(control group),the difference was statistically significant(P<0.05),and Guilu Erxian Jiao Decoction compared with cisplatin group reduced obviously(P<0.05),Guilu Erxian Jiao Decoction high dose group compared with cisplatin group reduction effect is not obvious,there was no statistically significant difference(P<0.05).3.4 Guilu Erxian Jiao Decoction + cisplatin,cisplatin +TGF-?1-siRNA can reduce the level of PD-1 m RNA expression in T cells,and more obvious than single use cisplatin or single use TGF-?1-si RNA,but it is not clear whether there is a synergistic effect.3.5 compared with cells in the control group,cisplatin group + TGF-?1-siRNA,Guilu Erxian Jiao Decoction group+ TGF-?1-si RNA group,cell control + TGF-?1-si RNA group can reduce PD-1 / PD-L1 / PD-L2 protein expression,the difference was statistically significant(P<0.05),cisplatin plus Guilu Erxian Jiao Decoction,cisplatin group,Guilu Erxian Jiao Decoction left PD-1 protein expression increased;The reduction of PD-1 protein expression in cisplatin +TGF-beta 1-si RNA group and TGF-?1-si RNA group was more significant than that in cell control +TGF-?1-si RNA group(P<0.05).There was no significant difference between cisplatin +TGF-?1-si RNA and TGF-?1-si RNA in the expression of PD-1 protein(P<0.05),but there was a significant difference between the two groups in influencing PD-L1 expression(P<0.05).4 Conclusion4.1 By inhibiting the apoptosis of T cells and reducing the PD-1+ expression of T lymphocytes around the tumor,Guilu Erxian Jiao Decoction can improve the cell immunity and inhibit the growth of tumor,improve the immune function of the tumor bearing mice,and achieve the purpose of anti tumor.4.2 Guilu Erxian Jiao Decoction can inhibit the apoptosis of CTLL-2 cells induced by cisplatin,thus reducing the immunosuppression induced by chemotherapy and protecting the cell immunity of the patients,which is beneficial to the treatment of tumor.