Fig 2: Effects of CHB-II-F on mRNA expression of NPY, AgRP, POMC, CART, OX1R, Ob-R,., and GHSR in the hypothalamus of the transplanted H22 hepatocellular carcinoma model mice receiving chemotherapy (x¯ ± s, n = 6). (A) NPY mRNA expression in different groups. (B) CART mRNA expression in different groups. (C) GHSR mRNA expression in different groups. (D) Ob-R mRNA expression in different groups. (E) POMC mRNA expression in different groups. (F) OX1R mRNA expression in different groups. (G) AgRP mRNA expression in different groups. (H) Gel electrophotogram of target gene NPY, AgRP, POMC, CART, OX1R, Ob-R, GHSR, and ß-actin amplification products of real-time fluorescent quantitation polymerase chain reaction (RTFQ-PCR). 1: AgRP; 2: NPY; 3: POMC; 4: CART; 5: OX1R; 6: Ob-R; 7: GHSR. (I) Gel electrophotogram of total RNA of tumor tissue. Randomly select three samples from each group and verify their integrity. 1–3: saline-treated controls (model); 4–6: 5-FU treated controls (0.02 g/kg); 7–9: YZXJC (0.78 g/kg); 10–12: CHB-II-F(H) (65 g/kg); 13–15: CHB-II-F(M) (32.5 g/kg); 16–18: CHB-II-F(L) (16.25 g/kg). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001 compared with the model (saline) group. # p < 0.05, ## p < 0.01 compared with the 5-FU group. ? p < 0.05 compared with the YZXJC group.

Fig 3: Effects of CHB-II-F on protein expression of NPY, AgRP, POMC, CART, OX1R, Ob-R, and GHSR in the hypothalamus of the transplanted H22 hepatocellular carcinoma model mice receiving chemotherapy detected by immunohistochemistry (x¯ ± s, n = 6). (A) Immunohistochemical pictures of NPY, AgRP, POMC, CART, OX1R, Ob-R, and GHSR protein expression in hypothalamus (× 400, scale bar = 200 µm). (B) NPY IOD values in different groups. (C) AgRP IOD values in different groups. (D) POMC IOD values in different groups. (E) CART IOD values in different groups. (F) OX1R IOD values in different groups. (G) Ob-R IOD values in different groups. (H) GHSR IOD values in different groups. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001 compared with the model (saline) group. # p < 0.05, ## p < 0.01 compared with the 5-FU group. ? p < 0.05 compared with the YZXJC group.

Fig 4: Effects of CHB-II-F on the content of NPY, OX1R, Ghrelin, Leptin, GAS, MTL, PGE2, and EGF in serum, SOD, and MDA in the jejunum of the transplanted H22 hepatocellular carcinoma model mice receiving chemotherapy (x¯ ± s, n = 6–8). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001 compared with the model (saline) group. # p < 0.05, ## p < 0.01 compared with the 5-FU group.

Fig 5: Colocalization of CB1R and OX1R in female mesocorticolimbic brain regions as a result of prolonged restraint stress and drug treatments. (A–C), CB1R-OX1R colocalization in the VTA (A,D), NAC (B,E), and MPFC (C,F) of 200 µL vehicle, 3 mg/kg SR141716A, 40 mg/kg YNT-185, and 3 mg/kg SR141716A + 40 mg/kg YNT-185-treated adult female C57BL/6 mice. On day 8, following the final sucrose preference measurement, mice were anesthetized and perfused in order to collect brain tissue for immunohistochemical analysis. Colocalization images were taken using a Zeiss LSM880 confocal microscope. Images were then analyzed on the FIJI extension of ImageJ (version 2.1.0). Colocalization data are expressed as Pearson’s correlation coefficients for n = 10 cells per treatment group randomly selected from three female mice. Means ± SEM were calculated, and a two-way ANOVA followed by a Tukey’s post-hoc analysis was used to determine statistical significance. @@@ p < 0.001 compared between drug treatments within 24 h RST. #/### p < 0.05/0.001 compared between No 24 h RST and 24 h RST within drug treatments. (D–F), Representative confocal microscopy images of CB1R-OX1R colocalization in the VTA (D), NAC (E), and MPFC (F) of females subjected to RST, which correspond to graphs (A–C), respectively. In the figure above “+” refers to with RST and “?“ refers to without RST.