The Effect Of IP10,CXCR1/2 Antagonist,combination With Cisplatin In Mice H22hepatocellular Carcinoma

Objective: Chemokines are a member of the cytokine family,mainly expressed in tumor cells and some non-immune cell surface,and have chemotactic and activating effects on neutrophils,lymphocytes,monocytes and other cells.Studies have shown that interleukin 8 were overexpressed in a variety of tumor cells,which not only contribute to neovascularization,but also to promote the invasion and metastasis of tumor cells.Interleukin-8(IL-8),also known as CXCL8,binds to its receptor CXCR1/2,induces neutrophils,lymphocytes and other cells aggregate to the injury site,activating intracellular downstream signaling pathway,and then play an important role in the occurrence,development,metastasis and other processes of tumor.iP10 is a non-biological activities CXCR1/CXCR2 competitive antagonist that hybridizes to IP10 and CXCL8 in our laboratory.It has a higher affinity for CXCR1/2 than CXCL-8,and competitively inhibits IL-8 and other chemokines binding with CXCR1/2,blocking the biological effects of a variety of cytokines to inhibit tumor angiogenesis,thereby affecting tumor growth and metastasis ability to play its anti-tumor effects.Cisplatin is a commonly used anti-tumor drugs,which has a significant effect in head and neck,lung,ovary,breast and other solid tumors.The anti-tumor effect of Cisplatin is significant,but it also produces toxic side effects,cisplatin-induced kidney damage is most serious.In this study,we will use iP10,cisplatin alone and the combination of the two treatment of liver cancer,to explore the development of iP10 on tumor development and cisplatin induced renal injury,for the clinical treatment of tumors to provide new ideasMethos:(1)BALB/c mice were inoculated 1�106 H22 cells subcutaneously(near the neck),observed the changes in the mental state of mice.On the 7th day after inoculation,the mice were randomly divided into 5 groups: control,iP10(before),iP10,DDP and iP10 + DDP group,eightteen mice per group.The mice of iP10 group and iP10 + DDP group were injected with iP10(500?g/kg of body weight;s.c.)subcutaneously.The other groups were treated with isovolumic saline at the same time..After1/2h,mice of DDP group and iP10 + DDP group were injected with cisplatin(12.5mg/kg;i.p.)once time.iP10 was injection every other day,the other groups injected with the same amount of saline.The tumor growth was monitored at any time and the tumor volume was recorded.(2)Specimen collection: On the 11 th day of inoculation of H22 hepatocellular carcinoma,8 mice were sacrificed in each group,and bilateral kidney and tumor tissues were dissected.On the 15 th day after inoculation,the remaining mice in each group were sacrificed.Eight mice in each group were sacrificed.Solid tumor tissues were dissected,weighed and photographed.(3)The tumor weight and tumor inhibition rate were calculated and statistically analyzed,and compared the difference of tumor growth rate in different groups.(4)H & E staining was used to detect the cell of tumor tissue.The expression of tumor-related proteins in tumor tissues were detected by immunohistochemistry and immunoblotting which was applied to observe the effects of different treatment on tumor growth.(5)Using H & E staining to assay the changes of glomeruli and renal tubules? The expression of inflammatory cytokines IL-1? and MIP-2 were detected by RT-PCR,and the myeloperoxidase activity was assaied by kits,which were used to investigate the effects of iP10 on renal injury induced by cisplatin.Results:1.The tumor growth in iP10 + DDP group was significantly lower than that in other groups,and the tumor growth rate in iP10 + DDP group was 78.60% � 0.09,which was significantly lower than that in other groups Was higher than that of DDP(38.69% � 0.08),iP10(44.08% � 0.22),iP10(before)(46.53% � 0.08)(P<0.05).2.The tumor histopathological results showed that there were different degrees of tumor necrosis in iP10(before)group,iP10 group,DDP group and DDP + iP10 group,and iP10 and cisplatin group showed necrosis.3.iP10 combined with cisplatin could decrease the expression of CXCR1,CXCR2,VEGF,EGFR and NF-?B in the tumor tissues by immunohistochemical method.4.Western blot results showed that the expression of VEGF,EGFR,MMP-2 and NF-?B protein in iP10 and cisplatin group were significantly lower than those in other groups,and the difference was statistically significant(P<0.05).5.The expression of IL-8 and VEGF in tumor tissue was detected by enzyme-linked immunosorbent assay(ELISA).The results showed that iP10 and cisplatin could significantly decrease the expression of IL-8 and VEGF in tumor microenvironment and inhibit the growth of tumor.6.From the renal histopathological examination results,iP10 can reduce the cisplatin treatment-induced renal tissue damage.7.The expression of IL-1? and MIP-2 in renal tissue was detected by RT-PCR.The results showed that iP10 combined with cisplatin could significantly decrease the expression of IL-1? and MIP-2 in renal tissue compared with DDP alone,Reduce the infiltration of neutrophils in renal tissue.8.Detection of myeloperoxidase(MPO)activity in renal tissue Compared with DDP alone,iP10 combined with cisplatin could decrease neutrophil infiltration and renal MPO activity in renal tissue.Conlusion:(1)iP10 combined with cisplatin significantly inhibited the growth of H22 tumors,and its inhibition was significantly stronger than the application of iP10 alone and DDP alone group,but the application of iP10 in advance compared with the normal application of iP10 was not statistically significant.(2)iP10 significantly reduced cisplatin-induced renal injury.Compared with the treatment group alone,iP10 combined with cisplatin reduced the expression of IL-1? and MIP-2,decreased the neutrophil infiltration and prolonged the expression of inflammatory cytokines in mice Survival time and improve the quality of life of mice.