关键词: 非酒精性脂肪性肝炎, 肝窦内皮细胞, 髓样上皮生殖酪氨酸激酶

Abstract: Objective To investigate the mechanism of myeloid-epithelial-reproductive tyrosine kinase (Mertk) expression in liver sinusoidal endothelial cells (LSECs) on non-alcoholic steatohepatitis (NASH) under a lipotoxic environment using plasmid transfection. Methods In the human liver sinusoidal endothelial cell line (TMNK-1), overexpression of Mertk (ov-Mertk) or knockdown of Mertk (sh-Mertk) was achieved through plasmid transfection. LSECs were treated with PA to simulate a high-fat environment, and were divided into 6 groups: palmitic acid (PA), bovine serum albumin (BSA, high-fat control group), ov-Mertk+PA, vector+PA (high-fat Mertk overexpression control group), sh-Mertk+PA, and sh-NC+PA (high-fat Mertk knockdown control group). Subsequently, LSECs from these 6 groups were co-cultured with the human hepatocellular carcinoma cell line (HepG2) using the Transwell method. The impact of co-culture on hepatocellular lipid metabolism, inflammatory response, and proliferation-related factors was assessed using quantitative polymerase chain reaction (qPCR) and Western Blot. Proliferation activity of HepG2 cells under co-culture conditions was evaluated using EdU assay. Independent sample t-tests were employed for data comparison between the two groups. Results (1) When LSECs of PA or PA+ov-Mertk were co-cultured with hepatocytes, the expression of fatty acid synthase (FAS), which promotes lipogenesis in hepatocytes, increased, while the expression of peroxisome proliferator activated receptor alpha (PPAR-α), which promotes lipolysis in hepatocytes, decreased. This condition was reversed when LSECs of PA+sh-Mertk co-cultured with hepatocytes (BSA vs. PA,FAS: 1.00±0.16 vs. 1.91±0.16, P=0.002,PPAR-α: 1.07±0.10 vs. 0.74±0.14, P=0.028;PA+vector vs. PA+ov-Mertk, FAS: 1.79±0.28 vs. 2.57±0.39, P=0.049,PPAR-α: 0.70±0.04 vs. 0.48±0.13, P=0.049;PA+sh-NC vs. PA+sh-Mertk, FAS: 1.93±0.29 vs. 1.37±0.16, P=0.045,PPAR-α: 0.70±0.12 vs. 0.95±0.06, P=0.035). (2) When LSECs of PA or PA+ov-Mertk were co-cultured with hepatocytes, the expression of interleukin-1β (IL-1β), which promotes inflammatory responses in hepatocytes, increased. However, the expression of IL-1β was reduced when LSECs of PA+sh-Mertk co-cultured with hepatocytes (BSA vs. PA, 1.17±0.16 vs. 2.51±0.23, P=0.001; PA+vector vs. PA+ov-Mertk, 2.37±0.15 vs. 3.81±0.33, P=0.002; PA+sh-NC vs. PA+sh-Mertk, 2.41±0.28 vs. 1.62±0.12, P=0.011). (3) When LSECs of PA or PA+ov-Mertk were co-cultured with hepatocytes, the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), which promote cell proliferation in hepatocytes, increased. However, the expression of CyclinD1 and PCNA was downregulated when LSECs of PA+sh-Mertk co-cultured with hepatocytes (BSA vs. PA, PCNA: 1.02±0.03 vs. 1.55±0.27, P=0.026,CyclinD1: 1.13±0.13 vs. 1.47±0.09, P=0.019;PA+vector vs. PA+ov-Mertk, PCNA: 1.57±0.20 vs. 2.04±0.16, P=0.032,CyclinD1: 1.29±0.44 vs. 2.32±0.35, P=0.034;PA+sh-NC vs. PA+sh-Mertk, PCNA: 1.51±0.30 vs. 0.98±0.13, P=0.048,CyclinD1: 1.42±0.09 vs. 1.11±0.16, P=0.045). (4) The EdU assay showed that the number of proliferating hepatocytes increased when hepatocytes were co-cultured with LSECs of PA or PA+ov-Mertk, while the number of proliferating hepatocytes decreased when they were co-cultured with LSECs of sh-Mertk [BSA: (12.86 ± 3.38) % vs. PA: (49.15 ± 4.5) %, P=0.0004;PA+vector: (42.15 ± 8.43) % vs. PA+ov-Mertk: (65.41 ± 0.66) %, P=0.009;PA+sh-NC: (48.19 ± 3.11) % vs. PA+sh-Mertk: (28.44 ± 1.96) %, P=0.001]. Conclusion In summary, our study found that overexpression of Mertk in LSECs could promote lipotoxic hepatocyte lipogenesis, inflammatory responses, and cell proliferation, thereby promoting NASH. Knockdown of Mertk in LSECs could serve as a target for NASH therapy.

Key words: NASH, LSECs, Mertk