Fig 1: Alcohol treatment induced liver fibrosis and activated the TLR4/MD-2–TNF-α pathway in model rats. In the alcohol-damaged liver of model rats, the molecules associated with liver fibrosis were upregulated, and the signaling pathway of liver fibrosis was activated. (A) Hematoxylin and eosin staining and (B) Masson staining were used to detect liver injury. The arrows indicate the portal area. Compared with that in the control group, the livers of rats in the EtOH group showed obvious pathological damage. (C) The levels of proteins related to liver fibrosis were detected by Western blotting, and data quantification was performed using ImageJ software; compared with that in the control group, the levels of proteins related to liver fibrosis in the EtOH group showed an obvious increase. (D–E) ELISA was used to determine the collagen-related markers (D) hydroxyproline and (E) ALT. The results showed that hydroxyproline and ALT levels significantly increased in the EtOH group. (F–G) The levels of TLR4, MD-2, and TNF-α were detected by (F) Western blotting and (G) RT-qPCR, and quantitation was performed using ImageJ software. The expression levels of TLR4, MD-2, and TNF-α were significantly increased in the EtOH group. The scale bar is 20 μm at high power and 80 μm at low power. **p<0.01, ***p<0.001, vs. the control group.
Fig 2: Dex protected hepatic I/R injury in vivo. The rat model of hepatic I/R was established. A, miR‐494 and JUND expression was detected using RT‐qPCR. B, The levels of ALT and AST in serum of rats were detected using ELISA. C, The rat liver sections were stained with HE staining. D, The rat liver sections were stained with immunohistochemical staining. E, The changes of JUND, p‐AKT, AKT, Nrf2 and NLRP3 were detected using Western blotting. N = 6. Data were presented as mean ± standard deviation and analysed using one‐way ANOVA, followed by Tukey's multiple comparison test, *** p < 0.001
Fig 3: Anti-inflammatory and antioxidant activities of puerarin in rats with bone graft defects. (A) Pro-inflammatory cytokines TNF-α, IL-1β, IL-17A, IL-6 and TGF-β1 in the serum of rats with bone grafts following treatment with puerarin or PBS. (B) Anti-inflammatory cytokines IL-2 and IL-10 in the puerarin and PBS groups in rats with bone grafts following treatment with puerarin or PBS. (C) Puerarin administration decreased serum ALT, AST, γ-GT, ALP, DBIL and TBIL levels in experimental rats. (D) Antioxidant activity in bone tissue of rats with bone graft defects following treatment with puerarin or PBS. Data are expressed as the mean ± standard deviation. Each experiment was repeated at least three times. Student's t-test was used to evaluate the statistical significance of differences between two groups. *P<0.05 and **P<0.01 vs. PBS. TNF, tumor necrosis factor; IL, interleukin; TGF, transforming growth factor; ALT, alanine transaminase; AST, glutamic oxaloacetic transaminase; γ-GT, γ-glutamyl transferase; ALP, alkaline phosphatase; DBIL, direct bilirubin; TBIL, total bilirubin.
Fig 4: Inhibition of TLR4/MD-2 can partially alleviate alcohol-induced liver fibrosis. Upon treatment with TLR4 and MD-2 antibodies, the expression of TLR4 and other molecules related to liver fibrosis was partially decreased, and the degree of injury was improved. (A–B) The levels of TLR4, MD-2, and TNF-α were detected by (A) RT-qPCR and (B) Western blot assays, which reveal that the antibodies of TLR4/MD-2 can partially inhibit the increase in TLR4, MD-2, and TNF-α induced by alcohol. (C) HE staining and (D) Masson staining were used to observe the degree of liver fibrosis in rats. The arrows indicate the portal area, and alcohol-induced liver fibrosis was partially relieved by inhibiting TLR4/MD-2. (E) Western blotting was used to determine the levels of proteins related to liver fibrosis, such as collagen I, α-SMA, and TIMP-1, and data were quantified using ImageJ software. The increase in these proteins is partially relieved by antibodies of TLR4/MD-2. (F–G) The expression of (F) hydroxyproline and (G) ALT is estimated by ELISA, and the increase in hydroxyproline and ALT induced by alcohol was partially relieved by antibodies of TLR4/MD-2. The scale bar is 20 μm at high power and 80 μm at low power. ***p<0.001 vs. the control group. ##p<0.01, ###p<0.001, vs. the EtOH group.
Fig 5: (A,B) Liver/tibia ratio, (C,D) plasma levels of aspartate aminotransferase (AST), (E,F) plasma levels of alanine transaminase (ALT), and (G,H) ratio of AST/ALT from sedentary (SED) or exercise-preconditioned (EX) male (left) and female (right) rats treated with doxorubicin (DOX) or saline. Data are presented as mean ± SEM. Two-way ANOVA differences are indicated below the graphs. Circles indicate individual data points. * = significant difference between groups (p < 0.05). ** = significant difference between groups (p < 0.01), *** = significant difference between groups (p < 0.001).
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