| Objective:1.Establishment of ALPPS animal model of tumor-bearing rats;2.Corosolic acid did not affect liver regeneration after ALPPS while inhibiting the effect of liver regeneration after ALPPS on tumor cell growth;3.The possible mechanism of corosolic acid reducing PD-1 expression and enhancing CD8+ T lymphocyte infiltration,inhibiting the growth of tumor cells induced by liver regeneration after ALPPS.Methods:1ml of Walker256 cell suspension（about 2 × 107 cells）was implanted in the medial posterior thigh of SD rats.After 10 days,the tumor with a diameter of about 1.5cm was seen.For tumor transplantation,an orthopedic puncture needle was used to puncture the left and right parts of the middle liver lobe and a tumor mass with a diameter of about 0.3 cm was transplanted.ALPPS was completed on the basis of the rat liver orthotopic tumor transplantation model,and the experiment was divided into 4 groups.Group I: control group;only laparotomy,hepatic ligament dissociation and abdominal closure were performed;Group II: orthotopic implantation group,based on the operation of Group I,tumors were implanted in the left and right middle lobes of the liver without ALPPS operation;Group III: implantation/ALPPS group,ALPPS operation was performed on the basis of the operation of Group II;group IV: transplantation/ALPPS/CA group,based on the operation of Group III,corosolic acid therapy was used after operation.Rats were sacrificed 15 days after operation,blood,tumor,liver,lung and kidney tissue samples were collected.The tumor mass and volume were measured,and liver and kidney functions were detected by biochemical automatic analyzer.Serum cytokine concentrations were detected by ELISA detection kit.CD8,CD86,CD206,CD31,Ki67,TGFβ,PD-1 and other indicators were detected and analyzed by immunehistochemistry（IHC）,and the liver,kidney and lung tissues were analyzed by HE staining.Results:（1）Animals on POD10 were sacrificed after anesthesia,and a gray-white mass with clear demarcation was seen in the liver,but there was no difference in volume and weight among the groups;animals were treated in the same way on POD15,and the average volumes of the II/III/IV groups were 394.8mm3,741.7mm3 and 371.6mm3.The average weights of the II/III/IV groups were 0.45 g,0.84 g and 0.41 g.It was found that the tumor demarcation was clear,and there was no intrahepatic and extrahepatic metastasis,and it was found that the FLR was significantly increased in the III and IV groups that underwent ALPPS operation.It was also found that the size and mass of tumors in group IV were significantly smaller than those in group III.（2）Comparing the liver proliferation rates（HRRs）in the right middle lobe（FLR） between different groups,the ALPPS operation group（Group III,Group IV）was compared with the non-operation group（Group I,Group II）,P<0.01,with a statistically significant difference.The comparison of the number of CD86+ Kupffer cells between the ALPPS-operated group and the non-operated group showed a statistical difference with P values <0.01,respectively.In addition,there was no statistical difference in the value of CD86+ Kupffer cells between Group III and Group IV groups subjected to ALPPS operation.There was also no statistical difference between the Group I and Group II groups that did not undergo ALPPS operation,and the P values were all >0.05.（3）Compared with the HRRs of Group II,the HRRs of Group III were significantly higher than those of Group II,with a P value of <0.01,which was statistically different.The number of Ki-67+ hepatocytes in Group III was significantly higher than that of Ki-67+ hepatocytes in Group II,P<0.01,with a statistical difference.In addition,the quality of Group III transplanted tumor was significantly higher than that of Group II transplanted tumor,and the difference was statistically significant,P<0.05.The number of Ki-67+ tumor cells in Group III was significantly higher than that of Ki-67+ tumor cells in Group II.The above experimental results showed that ALPPS accelerated the growth of FLR tumors while inducing liver regeneration.Further data analysis showed that the quality of Group IV tumors was significantly lower than that of Group III tumors,with a P value < 0.05,which was statistically significant.At the same time,the number of Group IV Ki-67+ tumor cells was significantly lower than that of Group III Ki-67+ tumor cells,P<0.05,with a statistical difference.（4）Morphological changes in the lungs,kidneys and livers of animals were found in groups II,III and IV: pulmonary interstitial edema,widened alveolar septum and inflammatory cell infiltration in the lungs;renal cortical arterioles and arterioles in the kidneys Para-inflammatory cell infiltration;inflammatory cell infiltration in the portal area of the liver and thickening of the GLISSION sheath.In addition,through the detection of liver and kidney function,it is suggested that ALPPS operation can damage liver function,but has no effect on kidney function,and the use of drugs does not affect kidney function.In addition,there was no statistical difference between the levels of Group III and Group IV liver function indexes and the changes in body weight,suggesting that the use of corosolic acid did not affect the changes in liver function and volume.（5）We used IHC technology to verify the polarizing effect of CA on tumor-associated macrophages（TAMs）,suggesting that corosolic acid can reduce M2-type macrophages and promote the conversion of M2-type macrophages to M1-type macrophages（Group IV Comparison of M1 and M2 within,and comparison of M2-type cells between Group IV and Group III）.（6）Tumor proliferation induced by ALPPS-induced liver regeneration,the number of TGF-β+ cells in Group III tumor was significantly higher than that in Group II（without ALPPS surgery）,the comparison between the two was P<0.01,which was statistically significant difference.In Group IV using CA,the number of TGF-β+ tumor cells was significantly lower than that of Group III TGF-β+ tumor cells,P<0.01.It is suggested that ALPPS induces tumor growth caused by liver regeneration and increases the expression of TGF-β in tumor cells.Corosolic acid can down-regulate the expression of TGF-β,which is similar to the expression of TGF-β in tumor cells.In addition,we detected plasma levels of TGF-β and VEGF by ELISA and found no difference between the groups.CD31 is a marker of vascular endothelial differentiation.The above experimental results suggest that ALPPS-induced liver regeneration accelerates tumor growth,which may be related to the increase of TGF-β and CD31 expression.The use of CA to inhibit tumor growth and downregulate the expression of TGF-β and CD31 in tumor tissue.（7）Comparing Group III and Group II PD-1+ cells found a statistical difference,P<0.01.Compared with Group III,Group IV was significantly lower than Group III,P<0.01.In addition,the analysis of the infiltration of CD8+ T cells in the tumor tissues of the three groups showed that the P values of Group II and Group III,and Group III and Group IV were all <0.01.Conclusion:（1）The tumor-bearing rat ALPPS model was successfully established;（2）The proliferation of hepatocytes induced by ALPPS is related to CD86+ Kupffer cells.（3）Corosolic acid inhibits tumor growth by inhibiting tumor cell proliferation.Corosolic acid has no effect on ALPPS-induced liver regeneration and can inhibit tumor proliferation caused by ALPPS-induced liver regeneration.（4）Corosolic acid does not aggravate the damage of other organs after ALPPS.（5）Corosolic acid（CA）can promote the conversion of M2 macrophages to M1 macrophages in tumors;down-regulate the expressions of TGF-β,CD31 and PD-1 in tumor tissues;and increase the infiltration of CD8+ lymphocytes.thereby exerting an anti-tumor effect. |
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