Non-alcoholic fatty liver disease combined with rheumatoid arthritis exacerbates liver fibrosis by stimulating co-localization of PTRF and TLR4 in rats - PubMed (original) (raw)
Non-alcoholic fatty liver disease combined with rheumatoid arthritis exacerbates liver fibrosis by stimulating co-localization of PTRF and TLR4 in rats
Shengpeng Zhang et al. Front Pharmacol. 2023.
Abstract
Rheumatoid arthritis (RA) has a high prevalence in patients with non-alcoholic fatty liver disease (NAFLD); however, the underlying mechanism is unclear. To address this, our study established a rat model with both NAFLD and RA by feeding a high-fat diet (HFD) and administering intradermal injection of Freund's complete adjuvant (FCA) with bovine type II collagen. Collagen-induced RA (CIA) was confirmed by hind paw swelling and histological examination. The histomorphological characteristics of NAFLD were evaluated by Masson's trichrome and hematoxylin-eosin staining. The development of NAFLD was further evaluated by measuring serum concentrations of triglyceride (TG), total cholesterol (T-CHO), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lipopolysaccharide (LPS). The results showed that HFD feeding exacerbated secondary inflammation in CIA rats, whereas FCA/bovine type II collagen injection increased serum levels of ALT, AST, TG, T-CHO, and LPS and exacerbated hepatic fibrosis in both normal and NAFLD rats. Interestingly, NAFLD + CIA significantly promoted the expression of PTRF, a caveolae structure protein involved in hepatic lipid metabolism and affecting downstream signaling of Toll-like receptor 4 (TLR4) and PI3K/Akt activation. High resolution confocal microscopy revealed increased PTRF and TLR4 co-localization in hepatic small vessels of NAFLD + CIA rats. AAV9-mediated PTRF knockdown inhibited TLR4 signaling and alleviated hepatic fibrosis in NAFLD + CIA rats. Together, these findings indicate that NAFLD combined with CIA causes synovial injury and enhances non-alcoholic fatty liver fibrosis in rats. PTRF could attenuate the symptoms of NAFLD + CIA likely by affecting TLR4/PTRF co-expression and downstream signaling.
Keywords: PI3K/AKT signaling; PTRF; TLR4; non-alcoholic fatty liver disease; polymerase I and transcript release factor; rheumatoid arthritis.
Copyright © 2023 Zhang, Zhu, Yuan, Cheng, Xu, Chen, Pan and Zheng.
Conflict of interest statement
Author WC was employed by the company Boster Biological Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
FIGURE 1
Effect of HFD and/or FCA/bovine type II collagen on synoviocyte proliferation in CIA and/or NAFLD rats. (A) Experimental protocol. (B) Images of the swelling of the secondary paw of CIA and NAFLD + CIA rats. (C) Degree of swelling of the non-injected hind paw (in mL) was measured. (D) Body weight changes in CIA rats with or without NAFLD. Values are presented as mean ± SD, n = 10. **p < 0.01 vs. Control group, ## p < 0.01 vs. NAFLD + CIA group, &&P < 0.01 vs. CIA group.
FIGURE 2
Histopathologic changes of the synovium in CIA and NAFLD rats. (A) In normal rats, synoviocytes were in monolayers (a) and articular cartilage was normal (b). (B) In CIA + NAFLD rats, serious proliferation of synoviocytes (a) and destruction of articular cartilage (b), with new blood vessels (or pannus) (c) and infiltration of inflammatory cells (d) were observed. (C) In NAFLD rats, there was slight destruction of articular cartilage (a), new blood vessels (b), pannus (c), and infiltration of inflammatory cells (d). (D) In CIA rats, destruction of articular cartilage (a), new blood vessels (b), pannus (c), and infiltration of inflammatory cells (d) were observed (b). Scale bar = 250 µm.
FIGURE 3
Expression of lipid- and liver function-related biomarkers in the serum of the rats in each group. (A, B) ALT and AST (related to liver injury) were detected to evaluate liver injury. (C, D) Serum TG and T-CHO levels as determined using respective kits. (E) Serum LPS levels were detected by ELISA kit. Data are presented as mean ± SD, n = 10. **p < 0.01 vs. control. ## p < 0.01 vs. NAFLD. &&P < 0.01 vs. CIA + NAFLD. &P < 0.05 vs. CIA + NAFLD.
FIGURE 4
Histopathologic changes of the liver in CIA and NAFLD rats. (A) Hepatic histopathology in rats determined by HE and Masson’s trichrome staining. Scale bar = 100 µm. (B) Quantitative analysis of Masson’s trichrome staining area. (C) The content of Hyp in the liver. Data are presented as mean ± SD, n = 10. **p < 0.01 vs. control, # p < 0.01 vs. CIA + NAFLD, ## p < 0.01 vs. CIA + NAFLD.
FIGURE 5
The expression of PTRF and the proteins in the PI3K/Akt signaling pathway in NAFLD/RA rats. (A) Western blot analysis of the expression of the proteins PTRF, PI3K (p85), p-PI3K, Akt, and p-Akt in the liver tissues of the rats. (B) Densitometric analysis of PTRF expression. GAPDH was used as the internal control. (C) Representative quantitative analyses of PI3K (p85), p-PI3K, Akt, and p-Akt levels. Data are reported as mean ± SD (n = 3). *p < 0.05 vs. control group, # p < 0.05 vs. CIA + NAFLD, &P < 0.01 vs. NAFLD.
FIGURE 6
The expression of PTRF and the proteins in the PI3K/Akt signaling pathway in NAFLD/RA rats. (A) Western blot analysis of the expression of the proteins PTRF, PI3K (p85), p-PI3K, Akt, and p-Akt in liver tissues. (B) Densitometric analysis of PTRF expression. GAPDH was used as the internal control. (C) Representative quantitative analyses of PI3K (p85), p-PI3K, Akt, and p-Akt levels. Data are reported as mean ± SD (n = 3). *p < 0.05 vs. control group, # p < 0.05 NAFLD + CIA/AAV-NC, & p < 0.05 NAFLD/AAV-NC.
FIGURE 7
(A) Co-localization of PTRF and TLR4 in merged confocal images representing overlays of PTRF (red), CD31 (green), and nuclear staining by DAPI (blue). Scale bar = 50 µm. (B) Left: Merged confocal images representing overlays of PTRF (red), TLR4 (green), and nuclear staining by DAPI (blue). Scale bar = 50 µm. Right: Quantitative data of each cell. Data are presented as mean ± SD, n = 5. **p < 0.01 vs. control, ## p < 0.01 vs. CIA + NAFLD/AAV-NC, && p < 0.01 vs. NAFLD/AAV-NC.
FIGURE 8
(A) Hepatic histopathology in rats determined by HE and Masson’s trichrome staining. Scale bar = 100 µm. (B) Quantitative analysis of Masson’s trichrome staining area. (C) The content of Hyp in the liver. Data are presented as mean ± SD, n = 5. **p < 0.01 vs. control, ## p < 0.01 vs. CIA + NAFLD/AAV-NC, && p < 0.01 vs. NAFLD/AAV-NC.
FIGURE 9
Left: Merged confocal images representing overlays of PTRF (red), α-SMA (green), and nuclear staining by DAPI (blue). Scale bar = 50 µm. Right: Quantitative data of each cell. Data are presented as mean ± SD, n = 3. **p < 0.01 vs. control, ## p < 0.01 vs. Control/AAV-PTRF-KD, && p < 0.01 vs. NAFLD + CIA/AAV-NC, & p < 0.05 vs. NAFLD + CIA/AAV-NC, △ p < 0.05 vs. NAFLD/AAV-NC.
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