Study On Antitumor Effect Of Compound Kushen Injection Combined With Cisplatin On Mice Bearing Lewis Lung Carcinoma

Among all cancers,lung cancer has the highest morbidity and mortality;it extremely threatens human life and health.It is difficult to detect lung cancer in the early stage.Once diagnosed,most patients have been in the middle and late stages,and the prognosis is very poor.Currently,surgery,chemotherapy,radiotherapy,and targeted therapy comprise the principal treatments for lung cancer.However,surgery for early stage cancer and chemotherapy for late cancer are still the principal treatment methods.Cisplatin has strong anti-tumor activity and broad anti-cancer spectrum and is widely used in clinical treatment of various cancers.However,its adverse reactions,such as nephrotoxicity,liver toxicity,and ototoxicity,have limited its clinical application.Therefore,combination therapy is recommended clinically to achieve effect-enhancing and toxicity-reducing.Compound kushen injection is made from Sophora flavescens and Baituling.It is used to treat cancer and has no obvious adverse effects.This project used CKI combined with DDP to achieve effect-enhancing and toxicity-reducing;and explored the antitumor effect and mechanism of the combination therapy to provide scientific theoretical bases for clinical medication.The main research contents are as follows:(1)40 Male C57BL/6 mice were assigned into four groups at random: Control group(Control),compound Kushen injection group(CKI),cisplatin group(DDP),combination therapy group(CKI+DDP),10 in each group.Lewis lung carcinoma mice model were established by subcutaneous injection of lewis cells.Antitumor effect of combination therapy was measured by tumor weight and tumor inhibition rate,etc.(2)ELISA assay was used to detect IL-4,IFN-? level in mice serum.(3)TUNEL staining was used to detect the apoptosis of tumor cells.Western blot assay was taken to detect the expression of apoptosis-related protein cleaved caspase-3.(4)ELISA assay was performed to measure the activity of hexokinase(HK),lactate dehydrogenase(LDH)in glycolytic pathway,the level of ATP(the end-product of energy metabolism)and lactate(the end-product of glycolysis)in tumor homogenate.Western blot assay was taken to detect the expression of glycolysis-related proteins PKM2 and PFKFB3.(5)The histopathological changes of liver and kidney of mice were observed by Hematoxylin and eosin(HE)staining to measure the protective effects of CKI on liver and kidney injuries induced by DDP.Through above researches,the following results were found:(1)General condition of mice: After subcutaneous inoculation of lewis lung carcinoma cells in mice for 7 days,solid tumors were tangible subcutaneously in the right axilla of mice.The feeding condition,mental state and reaction sensitivity of mice were not as good as before inoculation,and the fur of mice was no longer smooth and soft.With the growth of mice,the above situations further deteriorated.The condition of mice in the Control group was the most serious,and the condition of mice in the CKI group was the best.The conditions of mice were much similar in the DDP and CKI+DDP group,and were both better than Control group,but no better than CKI group.(2)Effects on body weight and effective body weight of mice: Compared with Control group,the body weight of mice executioned and effective body weight in CKI group showed no significant statistical difference(P>0.05),the body weight of mice executioned and effective body weight in DDP and CKI+DDP group exhibited significant statistical difference(P<0.001).compared with DDP group,thee body weight of mice executioned and effective body weight in CKI+DDP group had no significant statistical differences(P>0.05).(3)Effects on tumor weight and inhibition rate: Tumor weight in CKI,DDP and CKI+DDP group were respectively(5.93±1.43)g?(3.55±0.68)g?(1.92±0.67)g,they decreased significantly compared to Control group(P<0.01).compared with DDP group,tumor weight in CKI+DDP group significantly decreased(P<0.01).Tumor inhibition rate of CKI,DDP and CKI+DDP group were respectively 26.05%,55.68%,76.05%.According to Jin's formula,q=1.13,which suggested CKI combined with DDP showed an additive effect.(4)Effects on cytokines in mice serum: Compared with Control group,IFN-? and IL-4 level in serum of mice in CKI,DDP and CKI+DDP group showed no significant statistical differences(P>0.05).(5)Effects on the apoptosis of tumor cells: the apoptosis index of Control,CKI,DDP and CKI+DDP group were respectively 24.43%,38.83%,53.73%,68.33%.CKI,DDP and CKI+DDP significantly increased the apoptosis index(P <0.001).Compared to DDP group,CKI+DDP significantly increased the apoptosis index(P <0.01).(6)Effects on the expression of apoptosis-related protein: CKI and DDP alone had no significant effect on the expression of apoptosis-related protein Cleaved caspase-3(P >0.05),but CKI+DDP significantly increased the expression of Cleaved caspase-3(P <0.001).(7)Effect on glycolysis-related enzyme activities: Compared with Control group,HK and LDH enzyme activities in tumor tissues in CKI group had no significant statistical differences(P>0.05),HK and LDH enzyme activities in tumor tissues in DDP and CKI+DDP group were significantly downregulated(P<0.01).Compared with DDP group,HK and LDH enzyme activities in tumor tissues in CKI+DDP group exhibited no significant statistical differences(P>0.05).(8)Effects on glycolysis-related products: Compared with Control group,the ATP content in tumor tissues of mice were significantly reduced in CKI,DDP and CKI+DDP groups(P <0.001).The difference of ATP content between CKI + DDP group and DDP group was not statistically significant(P >0.05).The lactic acid content in tumor tissues in CKI,DDP and CKI+DDP groups had no significant statistical differences(P >0.05),compared to Control group.There was no significant difference in the lactic acid content between CKI + DDP group and DDP group(P >0.05).(9)Effect on glycolysis-related protein expression: CKI,DDP and CKI+DDP significantly downregulated the expression of PKM2 and PFKFB3 proteins in mouse tumor tissues(P <0.001).Compared with DDP group,the expression of PKM2 in mouse tumor tissues in CKI + DDP group showed no significant statistical differences(P >0.05);the expression of PFKFB3 in mouse tumor tissues in CKI + DDP group was upregulated significantly(P <0.001).(10)Effects on the histopathological morphology of liver in mice: Compared with the control group,the liver cells in the DDP group had a large number of degenerations,a large number of necrotic lesions and eosinophils.The hepatic cords were arranged very irregularly.There was a large amount of inflammation and severe necrosis in the liver lobes.The number of hepatocytes and hepatic cord arrangement were poorer than those of the CKI group,with a small amount of fat droplets,degeneration and focal necrosis of hepatocytes in the leaflets,a small number of eosinophils,and a small number of inflammatory cells around the liver.The degree of pathological damage in the CKI + DDP group was better than that in the DDP group.(11)Effects on the histopathological morphology of kidney in mice: Compared with the Control group,the glomerular volume of the DDP group differed.The tubular epithelial cells were irregularly arranged.A large number of inflammatory cells infiltrated in the renal tubules and tubulo-interstitial spaces,and the renal tubular epithelial cells were more granular and vacuole-like.Tubule epithelial cells were seen to shed more.More casts can be seen.The glomerular volume in CKI + DDP group was approximately normal,and the size was relatively consistent.There were more cells in the glomerulus than in the Control group,and the extracellular matrix was greater than that in the Control group.The tubular-interstitial structure was acceptable,with a small amount of infiltration of inflammatory cells,mild granular and vacuolar degeneration of tubular epithelial cells,and slight shedding of tubular epithelial cells.Less cast could be seen.The overall pathological damage degree of the CKI + DDP group was better than that of the DDP group.To sum up,the following conclusions can be drawn:(1)Compound Kushen injection can significantly enhance the antitumor effect of cisplatin,and the combination of the two drugs has an additive effect.In addition,compound Kushen Injection also has protective effects on liver and kidney damage caused by cisplatin.The combination therapy can achieve effectenhancing and toxicity-reducing.(2)Compound Kushen injection,cisplatin and combination therapy can all significantly inhibit tumor glycolysis by reducing the expression of PKM2,PFKFB3 protein,HK,LDH activity and ATP content in tumor tissues.But the combination of two drugs shows no synergistic effect on inhibiting glycolysis of tumor cells.(3)Compound Kushen injection,cisplatin and combination therapy can all significantly increase the apoptosis index of tumor cells,and combination therapy shows a synergistic effect on increasing the apoptosis index of tumor cells.Compound Kushen injection and cisplatin have no effect on the expression of cleaved caspase-3 protein in tumor tissues.However,the combination of two drugs can significantly increase the expression of cleaved caspase-3 protein in tumor tissues.In conclusion,the combination therapy can significantly enhance apoptosis of tumor cells,and the mechanism may be related to the increased expression of cleaved caspase-3 in tumor tissues.The combination of the two drugs has a synergistic effect on promoting tumor cell apoptosis.