Inhibitory Effect Of Astragalus Polysaccharide And Cisplatin On Recurrence And Metastasis Of Lewis Lung Cancer In Mice

Objective: The effects of Astragalus Polysaccharide on inhibiting Lewis lung cancer cells and metastasis of recurrent tumors in mice were observed in vivo and in vitro.Methods:(Ⅰ)In vitro experiments: LLC cells cultured in vitro were divided into control group,800 μg/m L APS treatment group,400 μg/m L APS treatment group,200 μg/m L APS treated group and 100 μg/m L APS treatment group,and each group was treated for 48 hours.The changes of cell migration,invasion and angiogenesis ability were detected by Transwell migration,invasion experiment and in vitro vascular imitation formation test.(Ⅱ)In vivo experiments: LLC was injected subcutaneously into palmula of left hind limb of C57BL/6J mice for 10 days as tumor-supply group.Other eighty C57BL/6J mice were randomly divided into 8 groups: model group,(50,100,200)μg/m L APS treatment groups,6 mg/kg DDP treatment group,3 mg/kg DDP combined(50,100,200)μg/m L APS treatment groups,10 animals per group.The single cell suspension of Lewis tumor from tumor-supply group was injected subcutaneously into palmula of left hind limb in each mouse.After 10 days,the tumor tissue from claw pad was resected,and the recurrent and metastatic mice model of postoperative lung cancer was established.From the next day,the mice in the treatment group were given 0.3m L drug for intraperitoneal injection.Observation indicators:(Ⅰ)In vitro experiments :(1)The ability of LLC cells induced neovascularization was observed via Vasculogenic mimicry formation assay.(2)The migration and invasion ability of LLC cells was detected by Transwell migration assay and Transwell Matrigel invasion assay.(Ⅱ)In vivo experiments:(1)We observed the general condition of each group of mice.(2)We calculated the weight of recurrent tumor and inhibition rate.(3)The pathological changes of recurrent tumor and lung in mice were examined by HE staining.(4)Immunohistochemistry staining was used to detect the protein expression of CD44,CD62 P and OPN in recurrent tumor tissues.(5)The protein expressions of MMP-2,MMP-9 and TIMP-2 in lung tissue were detected by Western blot.(6)The expressions of MMP-2,MMP-9 and TIMP-2 m RNA in lung tissue were detected by RT-PCR.Results:(1)Compared with the control group,the number of transmembrane cells in LLC cells in each APS treatment group was decreased(P<0.05),especially in the 800 μg/m L APS treatment group(P<0.01).(2)Compared with the control group,the number of neovascularization lumen in each APS treatment group decreased significantly(P<0.05),especially in 800 μg/m L APS treatment group(P<0.01).(3)There were multiple tumors with different sizes in the left hind limbs of mice in each group,which resulted in the limitation of their activities,especially in the model group.During the non-administration period,the body mass of mice in each group tended to change steadily,with no significant difference(P>0.05).There was a significant difference between the two groups in the administration period from tumor removal to execution(P<0.05).Compared with the model group,the inhibition rate of recurrence weight of mice in each treatment group was increased,especially in the DDP combined with 200 μg/m L APS treatment group(P<0.05).(4)HE analysis of recurrent tumors showed that the nucleus of the tumors was large and deep stained in the model group,and there was no necrosis around the tumors.Partial necrosis occurred in all treatment groups,especially in DDP group,followed by DDP combined with200 μg/m L APS group,with massive necrosis and nuclear fragmentation,pyknosis and necrotic cells.HE analysis of lung tissue showed that the alveolar epithelium of the blank group was intact without exudation,edema and bleeding.the alveolar epithelium of lung tissue in model group was partially absent,alveolar septum was significantly thicker,bleeding was serious,with a small amount of lymphocyte infiltration,and cell differentiation was low and high type.Compared with the model group,each treatment group improved in varying degrees.Especially in the combined group,alveolar epithelial deficiency was not significant,alveolar septum was slightly thickened,bleeding was reduced,fibrous tissue proliferation and lymphocyte-dominated inflammatory cell infiltration were found in some areas,and cell differentiation was higher and atypia was lower.(5)Immunohistochemical staining showed that the expression of CD44,CD62 P and OPN proteins in recurrent tumors in each treatment group was lower than that in model group.The DDP group was the most significant,and the expressions in DDP combined with(100,200)μg/m L APS group were decreased significantly(P<0.05).(6)Western blot analysis showed that the expression of MMP-2,MMP-9 and TIMP-2 in each treatment group was lower than that in the model group.The expression of MMP-2,MMP-9and TIMP-2 in the combined(high and medium)dose group was significantly lower than that in the model group.(7)Real-time fluorescence quantitative PCR showed that the gene expression levels of MMP-2,MMP-9 and TIMP-2 in each treatment group were lower than those in the model group(P<0.05).The combined(high and medium)dose group was more significant with statistical significance.Conclusion: APS can inhibit the migration and invasion of Lewis lung cancer cells to a certain extent,and reduce the ability to induce angiogenesis.APS combined with DDP can inhibit the recurrence and metastasis of lung cancer in mice and its mechanism may be related to decrease the expression of recurrent and metastasis-associated proteins including CD44,CD62 P,OPN,MMP-2,MMP-9 and TIMP-2.