Effects Of Astragalus Polysaccharides Combined With Cisplatin On The Growth Of Transplanted Lewis Lung Carcinoma In Mice Via Mitochondrial Mediated Pathway

Objective:By investigating the effects of Astragalus polysaccharides(APS)combi ned with Cisplatin on Lewis lung transplanted tumor in mice,on mitochondrial apopto sis related proteins like Caspase 3?Cyt C?Omi/Htr A2?Smac/Diablo?XIAP and on mitochondria ultrastructure,the mechanism of APS in restraining cancers was explored through mitochondrial mediated pathway.Methods:The C57BL/6J mice were divided randomly into nine groups(n=10 for each group),including a normal control group,a model group,three APS treated groups(50,100and 200?g/m L,respectively),a DDP treated group at a dose of 6mg/mL,and three DDP(3mg/kg)plus APS treated groups(50,100 and 200?g/mL,respectively).Except the mice in normal group,the rest mice were inoculated subcutaneously with LLC cells(1×10~7mL)in the right fore axilla.On the second day,the mice in the treatment group were given the drugs(0.3ml)by intraperitoneal injection.The mice in the DDP group were administrated once a week,while the rest were once a day.The mice in normal group and model group were given the same volume of NS for 20 days.All mice were killed on the 21st day.(1)We examined the general condition of mice during experiment.(2)We examined the weight and tumor weight of mice.(3)The tumor tissues were subjected to the histopathological examination via HE.(4)Immunohistochemical staining were used to detect the protein expressions of Caspase 3,Cyt C,Omi/HtrA2,XIAP and Smac/Diablo.(5)The expression of apoptosis protein including Caspase 3 and Smac/Diablo in tumor cells were detected by Western blot.The mitochondrial morphology of tumor cells were observed by transmission electron microscope.Results:(1)Compared with the model group,the weight of DDP group was decreased significantly(p<0.05).The tumor weight of the mice in the 3 mg/kg DDP combined APS groups(100 and 200?g/mL)were reduced significantly(p<0.05).We found that the weight of tumor was reduced,compared with the DDP group,in the mice of the 3 mg/kg DDP combined(100 and 200)?g/mL APS groups(p<0.05).(2)HE staining of lung tissue showed that the alveolar structure of the model group and DDP group have disappeared.The lung tissues in the 3 mg/kg DDP combined(100 and200)?g/mL APS groups were relatively complete,with a slight thickening of the alveolar diaphragm,and lymphocyte infiltration.HE staining of tumor tissue showed that tumor cells in the model group had large nuclei,stained deeply and there was no necrosis around.The tumor cells in DDP group were stained deeply,and some tumor cells were necrotic.200?g/mL APS combined 3 mg/kg DDP group showed large necrosis,nuclear fragmentation and consolidation.(3)Immunohistochemistry showed that compared with those of model group,Caspase 3 in tumor tissues,Cyt C,Smac/Diablo,Omi/HtrA2 protein expression increased significantly in treatment groups.Compared with DDP group,expression difference of the above factors in3 mg/kg DDP combined with 200?g/mL APS group was the most significant(p<0.05 or p<0.01).The XIAP protein was positive and high expression in the model group.Compared with the model and DDP groups,the XIAP protein expression difference in the 3 mg/kg DDP combined(100 and 200)?g/mL APS groups was significant(p<0.05 or p<0.01).(4)TheexpressionofapoptosisproteinincludingCaspase3and Smac/Diablo in tumor cells were detected by Western blot.(5)Transmission electron microscopy(TEM)showed that in model group,mitochondria structure are clear and visible;there was mitochondria crest sample structure,and no swelling,pyknosis,neat rows,the membrane appearance was complete with no mitochondria cavity structure,we observed that the chromatin distribution was uniform,the nuclei were large and the cytoplasm was small.In the low,middle and high dose APS-treated groups,we observed nucleus chromatin aggregation,large nucleus,small cytoplasm mitochondrial swelling in cytoplasm.In the DDP group,there was mitochondrial swelling in the cytoplasm,disorder of mitochondrial arrangement,large chromatin in nucleus and vacuole in cytoplasm.In the 3 mg/kg DDP combined 100 and 200?g/mL APS groups,we found that the nucleus chromatin gathered,some cells appeared apoptosis,less cytoplasm,mitochondria swelling in the cytoplasm,mitochondria pyknosis,crest structure disorder and disappear,the membrane was broken and cavity formation can be seen in the mitochondria.Conclusion:These results indicated that the APS combined DDP had a certain inhibitory effect on Lewis lung carcinoma.APS and DDP combination chemotherapy can inhibit the growth of Lewis lung cancer cells in mice and its mechanism may be associated with increased mitochondrial apoptosis pathway,increased expression of prot eins Caspase 3,Cyt C,Smac/Diablo,Omi/HtrA2 and decreased XIAP expression.