Effect And Regulation Mechanism Of Peritoneal Macrophages Function Change In Gastric Cancer Peritoneal Metastasis

Background: Peritoneal metastasis of gastric cancer is the leading cause of death in postoperation gastric cancer patients. Whether laparoscopy with CO₂ pneumoperitoneum affects the peritoneal metastasis of gastric cancer is a pressing question. Peritoneal macrophages are the most important peritoneal immune cells. The role and mechanism of peritoneal macrophages in the peritoneal metastasis of gastric cancer remain unclear. The study aim is(1)to investigate the functional changes of peritoneal macrophages and their effects and possible mechanism in the peritoneal metastasis of gastric cancer. (2)to explore TGF-β1 induced peritoneal macrophages function change, and the possible regulation mechanism of peritoneal macrophages change.(3)to investigate the change in peritoneal macrophages function in gastric cancer in the CO₂ pneumoperitoneum environment, as well as its effect on the peritoneal metastasis of gastric cancer.Method and Results:Part 1:The role of peritoneal macrophages in the peritoneal metastasis of gastric cancerMethod :The animals were divided into three groups(n=12):group A,normal mice,control group;group B,transplantation model of forestomach carcinoma mice ;group C,an anti-TGF-β1 antibody group, transplantation model of forestomach carcinoma mice which received an intraperitoneal injection of the anti-TGF-β1 antibody. Peritoneal macrophages were isolated and purified from three groups mice after ten days. The TGF-β1 level was examined in the supernatant of peritoneal fluid.The changes in the phagocytosis and the secretion of NO, TNF-α, IL-10, and VEGF, which were isolated from peritoneal macrophages. The peritoneal metastasis of forestomach carcinoma were observed between group B and group C. Results: The TGF-β1 concentration of group A ,group B ,group C was 17.56±5.02 pg/ml,224.04±12.36 pg/ml,50.37±10.82 pg/ml separately,compared with the TGF-β1 concentration of group A ,group B and group C were significantly increased,group C were significantly reduced compared with group B. Compared with group A, the peritoneal macrophages significantly decreased phagocytosis and levels of NO,TNF-αsecretion at 12h,24 h, 48 h, and 72 h (P < 0.05), significantly increased IL-10 and VEGF secretion in group B and group C (P < 0.05). Compared with group B,the peritoneal macrophages of phagocytosis and levels of NO and TNF-αsecretion significantly increased,IL-10 and VEGF secretion significantly decreased in group C.The peritoneal metastasis rate was 41.66%(5/12) of group C, while it was 100% (12/12) in group B. The implantation nodule weight was 0.52±0.24 g in group C, which was significantly lower than the weight from group B (1.08±0.17 g).Part 2: The role and mechanism of TGF-β1-induced peritoneal macrophages in the peritoneal metastasis of gastric cancerMethods Culture mouse forestomach cells (MFC), The concentration of MFC supernatant TGF-β1 were measured using ELISA kits. Immunohistochemical observed TGF-β1 protein expression. Separation and purification mouse peritoneal macrophage, using recombinant TGF -β1 stimulate mouse peritoneal macrophage, peritoneal macrophage phagocytosis detection, the concentration of MFC supernatant NO, IL -10, TNF-αand VEGF secretion were measured using kits. Immunohistochemical and Western blotting observed peritoneal macrophages SMAD2/3, SMAD4, SMAD7, VEGF expression. Results The protein expression of TGF-β1 is high, TGF-β1 concentration of 1 x 10⁶ / ml MFC supernatant cultured for 48h was 107.56±4.82 pg/ml. Using 100pg/ml of TGF - beta 1 stimulate mouse peritoneal macrophage, the phagocytosis,NO and TNF-αsecretion was reduced significantly after cultured for 24h, 48h, 72h control with the normal mouse peritoneal macrophage(P < 0.05), IL - 10, VEGF was increased significantly (P < 0.05). Immunohistochemical results show that TGF -β1 stimulation group, SMAD2/3, SMAD4, SMAD7 and VEGF expression significantly strengthened cultured for 24h, 48h, 72h control with the normal mouse peritoneal macrophage.Western -blotting also made similar results.Part 3: Effects of CO₂ pneumoperitoneum on peritoneal macrophage function and peritoneal metastasis in mice with gastric cancerMethods: An orthotopic transplantation model of murine forestomach carcinoma was established using the mouse line. The mice bearing tumors were randomly divided into six groups (n = 30): anesthesia alone, laparotomy, mini-laparotomy, and 2 mmHg,4 mmHg,6 mmHg CO₂ insufflation group. Peritoneal macrophages were collected from 6 mice in each group and cultured. The phagocytosis by macrophages and the levels of NO, TNF-α,IL-10,VEGF produced by macrophages were measured after 12, 24, 48, and 72 h of culture. The remaining mice were observed after two weeks for the rate of peritoneal metastasis of forestomach carcinoma cells and the total weight of implanted nodules. Results: The uptake of neutral red by peritoneal macrophages and the levels of NO, TNF-α,IL-10,VEGF secreted by peritoneal macrophages in the laparotomy group and mini-laparotomy group after 12h of culture were all significantly higher than those in the anesthesia alone group(P<0.05). The corresponding levels in the 2 mmHg CO₂ insufflation group after 12 h were significantly lower than those in the laparotomy group (P<0.05). The corresponding levels in the 4 mmHg CO₂ insufflation group after 12 h were all significantly lower than those in the anesthesia alone group and laparotomy group (P<0.05). The corresponding levels in the 6 mmHg CO₂ insufflation group after 12h,24h were all significantly lower than those in the anesthesia alone and laparotomy group (P<0.05). The rate of peritoneal metastasis in 6 mmHg CO₂ group was significant higher than the laparotomy group(75% to 50%,P <0.05).The total weight of implanted nodules of mouse forestomach carcinoma were no significant differences in the 2 mmHg, 4 mmHg,6 mmHg CO₂ group and the laparotomy group (P>0.05).Conclusion(1)The phagocytosis and NO and TNF-αsecretion by peritoneal macrophages decreased and the secretion of IL-10 and VEGF increased in gastric cancer. Peritoneal macrophage function changes is closely related to peritoneal metastasis of gastric cancer.(2)TGF–β1 suppresses the peritoneal macrophages phagocytosis and secretion of NO and TNF alpha, promotes the secretion of IL– 10 and VEGF, play an important role in the peritoneal metastasis process of MFC, may be connected with the activating of peritoneal macrophage TGF-β1/SMADS signaling pathways.(3)Low pressure CO₂ pneumoperitoneum (2 mmHg, 4 mmHg)neither significantly changes the phagocytosis and cytokine secretion functions of peritoneal macrophages in gastric cancer-bearing mice nor significantly promotes peritoneal metastasis of gastric cancer,but high pressure CO₂ pneumoperitoneum (6 mmHg)can inhibit the phagocytosis and cytokine secretion NO, TNF-αfunctions of peritoneal macrophages in gastric cancer-bearing mice in long time and promote peritoneal metastasis of gastric cancer. (4)Functional changes of peritoneal macrophages in gastric carcinomas are an important factor leading to the peritoneal metastasis of gastric cancer.