Objective:To explore the influence of fentanyl on sorafenib sensitivity in the treatment of hepatocellular carcinoma (HCC) pain.Methods:Adopting a prospective research method, all laboratory tests were conducted in the Central Laboratory of Xinhua Hospital, Dalian University from August 2021 to August 2023. CCK-8 method, clone formation assay and cell counting under light microscope were used to detect cell proliferation of the human liver cancer cell line HepG2. LysoTracker, monodansyl cadaverine and transmission electron microscopy (TEM) were used to detect the levels of autophagy in fentanyl-induced HCC cells. The level of reactive oxygen species was measured using the fluorescent dye, 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Protein immunoblotting test was applied to detect the protein level of upstream protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. The effects of the antitumor efficacy of fentanyl combined with sorafenib were evaluated by the HCC tumor-forming experiment in nude mice. Fifteen female BALB/c nude mice (6-week-old) were divided into control group, sorafenib group and sorafenib + fentanyl group by random digits table method with 5 mice each. The nude mice in three groups were subcutaneously injected at left axilla HepG2 cells (4 ×10 6). The nude mice in control group, sorafenib group and sorafenib + fentanyl group were respectively treated with injection of 1 ml of 0.9% sodium chloride, oral sorafenib with 20 mg/kg, oral sorafenib with 20 mg/kg combined with intravenous injection fentanyl with 0.05 mg/kg every other day for 3 consecutive weeks. The tumor volume was measured at the 4th、8th、12th、16th、20th and 24th day. The nude mice were sacrificed after tumors formed and the tumor tissues were collected and weighed. The tumor tissue was checked by the immunohistochemical staining assay. Results:The CCK-8 and clone formation assay results showed that fentanyl reduced the antitumor effect of sorafenib in HCC. Fentanyl upregulated the expression of autophagy-related proteins Beclin-1 and LC3, and immunofluorescence and transmission electron microscopy showed that fentanyl could increase the levels of autophagy in HCC cells. DCFH-DA staining showed that fentanyl increased reactive oxygen species production, and Protein immunoblotting test result showed that fentanyl reduced the levels of phosphorylated protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR). The autophagy inhibitor 3-methyladenine (3-MA) could block the protective autophagy induced by fentanyl, and then reduce the adverse effects of fentanyl on sorafenib. There was no statistical difference in tumor volume at 4th, 8th and 12th day among three groups ( P>0.05); the tumor volume 16th, 20th and 24th and weight in fentanyl + sorafenib group and sorafenib group were significantly lower than those in control group: (275.00 ± 11.58) and (174.00 ± 91.42) mm 3 (307.40 ± 81.39) mm 3, (701.00 ± 105.08) and (563.60 ± 89.59) mm 3 vs. (855.20 ± 68.71) mm 3, (971.60 ± 79.87) and (691.80 ± 11.17) mm 3 vs. (1 177.20 ± 105.79) mm 3, (705.00 ± 35.50) and (540.20 ± 80.76) mg vs. (1 118.40 ± 76.81) mg, the indexes in sorafenib group were significantly lower than those in fentanyl + sorafenib group, and there were statistical differences ( P<0.05). The immunohistochemical staining assay result showed that Ki67 and LC3 positive cells (brown cells) in fentanyl + sorafenib group were significantly more than those in sorafenib group. Conclusions:Fentanyl reduces the suppressive effect of sorafenib on HCC tumor growth by inducing protective autophagy, which could be weakened by adding autophagy inhibitors.