[Lactate promotes HMGB1 phosphorylation and release via Akt signaling pathway in gastric cancer cells HGC-27].

Objective: To investigate the molecular mechanism of how lactate induces high mobility group box 1 (HMGB1) release. Methods: Gastric cancer HGC-27 cells were divided into the control group and the lactate group (The cells were treated with lactate for 6 h). The level of HMGB1 in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA), the localization of HMGB1 was detected using laser confocal microscopy, and the nuclear translocation of HMGB1 was detected using the nucleoplasmic separation assay. The phosphorylation and acetylation levels of HMGB1 were determined by co-immunoprecipitation, and Western blot was used to measure the phosphorylation of Akt and protein kinase C (PKC). HGC-27 cells were first treated with lactate and LY294002, the inhibitor of Akt, and then the phosphorylation of HMGB1 and Akt was analyzed by co-immunoprecipitation and Western blot, respectively. The localization of HMGB1 in cells was detected by laser confocal microscopy. EdU and Transwell assays were used to detect the proliferation and migration abilities of HGC-27 cells, respectively. HGC-27 cells were then injected into the BALB/C null mice for subcutaneous tumor implantation. Mice in the lactate group were intraperitoneally injected with lactate (0.2 g/kg/2 d), while those in the control group were intraperitoneally injected with an equal amount of PBS for 20 consecutive days. ELISA was used to detect the HMGB1 levels in the blood samples taken from the medial canthus vein of the mice, while co-immunoprecipitation and Western blot were used to detect the phosphorylation of HMGB1 and Akt in tumor tissue proteins, respectively. Results: The release levels of HMGB1 in the lactate group were (2 995.00±660.91) pg/ml and (696.33±22.03) pg/ml, after lactate treatment for 6 h and 12 h, respectively, both higher than those in the control group (485.00±105.83) pg/ml (P<0.001 and P=0.028, respectively). After lactate treatment for 6 h, the relative expression of HMGB1 protein in the cytoplasm of HGC-27 cells was 1.13±0.09, higher than that of the control group (0.83±0.07, P=0.001), while the relative expression of HMGB1 in the nucleus was 0.79±0.06, lower than that of the control group (1.07±0.06, P=0.007). The phosphorylation level of HMGB1 reached 1.41±0.09, which was higher than that of the control group (0.97±0.10, P=0.031). The phosphorylation level of Akt was 11.16±0.06, higher than that of the control group (0.91±0.022, P=0.002). The phosphorylation level and nuclear translocation of HMGB1 induced by lactate decreased obviously after Akt inhibition; the proliferation and migration abilities induced by lactate were also obviously inhibited after Akt inhibition. In vivo, the HMGB1 level in the peripheral blood was (1 280.70±389.66) pg/ml in the lactate group, which was obviously higher than that in the control group (595.11±44.75) pg/ml (P=0.008), and the phosphorylation levels of HMGB1 and Akt in tumor tissues in the lactate group were obviously enhanced compared with the control group. Conclusion: Lactate induces HMGB1 release through enhancing HMGB1 phosphorylation via the Akt signaling pathway.目的： 探讨乳酸诱导胃癌细胞高迁移率族蛋白盒1(HMGB1)释放的分子机制。 方法： 将胃癌HGC-27细胞分为对照组和乳酸处理6 h组，通过酶联免疫吸附试验(ELISA)检测细胞培养液中HMGB1的水平，激光共聚焦和核质分离实验分别检测HMGB1的胞内定位及核转位，免疫共沉淀检测HMGB1磷酸化和乙酰化，Western blot检测Akt和蛋白激酶C的磷酸化。以Akt抑制剂LY294002与乳酸联合作用HGC-27细胞后，采用免疫共沉淀和Western blot分别检测HMGB1和Akt的磷酸化，激光共聚焦检测HMGB1的胞内定位，EdU实验检测细胞的增殖能力，Transwell实验检测细胞的迁移能力。将HGC-27细胞注射于BALB/C裸鼠腋部皮下建立移植瘤模型。乳酸组小鼠隔天腹腔注射乳酸(0.2 g/kg)，对照组小鼠隔天腹腔注射等量PBS，连续20 d。ELISA法检测裸鼠内眦静脉血HMGB1含量，免疫共沉淀和Western blot检测肿瘤组织中HMGB1和Akt的磷酸化。 结果： 乳酸作用6和12 h，HMGB1的释放量分别为(2 995.00±660.91)pg/ml和(696.33±22.03)pg/ml，均高于对照组[(485.00±105.83)pg/ml，P值分别为<0.001和0.028]。乳酸处理6 h后，HGC-27细胞的细胞质中HMGB1蛋白的相对表达量为1.13±0.09，高于对照组(0.83±0.07，P＝0.001)，而细胞核中HMGB1的相对表达量为0.79±0.06，低于对照组(1.07±0.06，P＝0.007)；HMGB1的磷酸化水平达1.41±0.09，高于对照组(0.97±0.10，P＝0.031)；Akt的磷酸化水平为11.16±0.06，高于对照组(0.91±0.022，P＝0.002)。抑制Akt活化后，乳酸诱导的HMGB1磷酸化水平降低，HMGB1核转位明显减少，细胞增殖和迁移能力亦下降。体内实验显示，乳酸组小鼠外周血中HMGB1的含量为(1 280.70±389.66)pg/ml，高于对照组[(595.11±44.75)pg/ml，P＝0.008]，肿瘤组织中HMGB1和Akt的磷酸化水平明显增强。 结论： 乳酸通过激活Akt信号通路增强胃癌细胞中HMGB1的磷酸化，促进HMGB1释放。.