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. 2024 May 14;22(1):457.
doi: 10.1186/s12967-024-05228-1.

Resveratrol attenuates inflammation and fibrosis in rheumatoid arthritis-associated interstitial lung disease via the AKT/TMEM175 pathway

Affiliations

Resveratrol attenuates inflammation and fibrosis in rheumatoid arthritis-associated interstitial lung disease via the AKT/TMEM175 pathway

Nannan Liu et al. J Transl Med. .

Abstract

Background and purpose: Interstitial lung disease (ILD) represents a significant complication of rheumatoid arthritis (RA) that lacks effective treatment options. This study aimed to investigate the intrinsic mechanism by which resveratrol attenuates rheumatoid arthritis complicated with interstitial lung disease through the AKT/TMEM175 pathway.

Methods: We established an arthritis model by combining chicken type II collagen and complete Freund's adjuvant. Resveratrol treatment was administered via tube feeding for 10 days. Pathological changes in both the joints and lungs were evaluated using HE and Masson staining techniques. Protein expression of TGF-β1, AKT, and TMEM175 was examined in lung tissue. MRC-5 cells were stimulated using IL-1β in combination with TGF-β1 as an in vitro model of RA-ILD, and agonists of AKT, metabolic inhibitors, and SiRNA of TMEM175 were used to explore the regulation and mechanism of action of resveratrol RA-ILD.

Results: Resveratrol mitigates fibrosis in rheumatoid arthritis-associated interstitial lung disease and reduces oxidative stress and inflammation in RA-ILD. Furthermore, resveratrol restored cellular autophagy. When combined with the in vitro model, it was further demonstrated that resveratrol could suppress TGF-β1 expression, and reduce AKT metamorphic activation, consequently inhibiting the opening of AKT/MEM175 ion channels. This, in turn, lowers lysosomal pH and enhances the fusion of autophagosomes with lysosomes, ultimately ameliorating the progression of RA-ILD.

Conclusion: In this study, we demonstrated that resveratrol restores autophagic flux through the AKT/MEM175 pathway to attenuate inflammation as well as fibrosis in RA-ILD by combining in vivo and in vitro experiments. It further provides a theoretical basis for the selection of therapeutic targets for RA-ILD.

Keywords: Autophagy; RA-ILD; Resveratrol; TMEM175.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Resveratrol alleviates RA-ILD fibrosis. A 8-week-old male C57BL/6 mice were randomly divided into 3 groups of 5–8 mice each: (I) Ctrl (control), (II) CIA (collagen-induced arthritis model), and (III) CIA + Res (CIA and resveratrol-treated model). HE staining of mouse joints. B HE staining of mouse lungs. C Masson staining of mouse lung. D Mouse lung tissue qRT-PCR assay E Mouse lung tissue hydroxyproline assay. F Immunohistochemical detection of CollagenI and TGF-β1 protein expression in mouse lung tissue. G Western blot detection of CollagenI protein expression in mouse lung tissue. H Western blot for TGF-β1 protein expression in mouse lung tissue. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 2
Fig. 2
Resveratrol targets TGF-β1 to alleviate RA-ILD progression. A Molecular docking modeling of resveratrol with TGF-β1. B Molecular dynamics simulation verified the binding ability between resveratrol and TGF-β1 protein. C An in vitro cell model was constructed using TGF-β1 + IL-1β-induced MRC-5 cells, and Western blot detected Collagen I protein expression in the resveratrol concentration gradient-treated MRC-5 cell model. D Western blot detection of TGF-β1 protein expression in MRC-5 cell model after resveratrol treatment. E Immunofluorescence detection of TGF-β1 protein expression in MRC-5 cell model after resveratrol treatment. F CCK8 detection of viability of MRC-5 cell model after resveratrol treatment. G EDU detection of cell cycle in MRC-5 cell model after resveratrol treatment. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 3
Fig. 3
Resveratrol attenuates inflammation and oxidative stress in RA-ILD. A Immunohistochemical detection of TNF-α and IL-1β protein expression in lung tissue. B Western blotting was used to detect P-NFKB, NFKB, TNF-α, and IL-1β protein expression in lung tissue. C qRT-PCR for TNF-α, IL-1β RNA expression in lung tissue. D MDA detects lung tissue oxidation levels. E Western blotting to detect SOD1 and SOD2 protein expression in lung tissue. F Western blotting to detect P-NFKB, NFKB, IL-1β protein expression in MRC-5 cell model after resveratrol treatment. G Western blotting to detect HIF-1α, SOD2 protein expression in the MRC-5 cell model after resveratrol treatment. H DCFH-DA and JC-1 were used to detect the levels of ROS and membrane potential in the MRC-5 cell model after resveratrol treatment, respectively. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 4
Fig. 4
Resveratrol restores autophagy abnormalities in RA-ILD. A Immunohistochemical detection of LC3-II, P62, and LAMP2 protein expression in lung tissue. B Western blotting for LC3-II, P62, and LAMP2 protein expression in lung tissue. C Western blotting to detect P-mTOR, mTOR, P-ULK1, and ULK1 protein expression in lung tissues. D Western blotting to detect Beclin1, Atg5, and Bnip3 protein expression in lung tissue. E MDC detection of autophagic vesicle changes in the MRC-5 cell model after resveratrol treatment. F Immunofluorescence detection of LC3-II, P62, and LAMP2 protein expression in MRC-5 cell model after resveratrol treatment. G Western blot detection of LC3-II, P62 protein expression in MRC-5 cell model after resveratrol treatment. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 5
Fig. 5
Resveratrol attenuates RA-ILD by promoting autophagic lysosomal fusion to restore autophagic flux. A qRT-PCR for RNA expression of PKC-A, PKC-B, PKC-D, GSK3B and TFEB in lung tissue. B qRT-PCR for lung tissue RAB7A, TBC1D15, FIS1 RNA expression. C qRT-PCR to detect lung tissue VAMP8, SNAP29, STX17 RNA expression. D qRT-PCR to detect VATPASE, ATP6AP1, ATP6AP2 RNA expression in lung tissues. E Western blotting to detect P62, LC3-II protein expression in MRC-5 cells after CQ concentration gradient treatment with resveratrol. F Western blotting detection of P62, LC3-II protein expression in MRC-5 cells after BAFA1 concentration gradient treatment with resveratrol. G Western blotting detection of P62, LC3-II protein expression in MRC-5 cells after resveratrol treatment with MG132 concentration gradient. H Western blotting detection of P62, LC3-II, Collagen I protein expression in MRC-5 cells after resveratrol treatment with CQ, BAFA1, MG132. I mCherry-GFP-LC3 detection in MRC-5 cells after CQ treatment with resveratrol. J Western blot detection of P62, LC3B protein expression in CQ + TGF-β1 + IL-1β-treated MRC-5 cells. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 6
Fig. 6
TMEM175 promotes RA-ILD progression by inhibiting autophagic lysosomal fusion. A qRT-PCR to detect TMEM175 RNA expression in lung tissues. B Western blotting to detect TMEM175 protein expression in lung tissues. C Immunohistochemistry for TMEM175 protein expression in lung tissue. D Western blotting to detect TMEM175 protein expression in MRC-5 cells after resveratrol treatment. E Immunofluorescence to measure TMEM175 protein expression in MRC-5 cells after resveratrol treatment. F Western blotting to detect TMEM175 protein expression after interference. G mCherry-GFP-LC3 detection of autophagy changes after interference with TMEM175. H Lyso-Tracker Red staining to detect lysosomal changes after TMEM175 interference. I Western blotting detection of LC3-II, P62, and Collagen I protein expression after interference with TMEM175. J EDU detection of cell proliferation after interference with TMEM175. K Flow cytometry detection of cell cycle after interference with TMEM175. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 7
Fig. 7
AKT assists TMEM175 in the regulation of autophagic lysosomal fusion. A Western blotting to detect P-AKT and AKT protein expression in lung tissue. B Immunohistochemistry to detect AKT protein expression in lung tissue. C Western blotting to detect P-AKT, AKT protein expression in MRC-5 cells after resveratrol treatment. D Immunofluorescence detection of AKT protein expression in MRC-5 cells after resveratrol treatment. E Western blotting to detect P-AKT protein expression in SC79 concentration gradient-treated MRC-5 cells. F Western blotting detection of P-AKT protein expression in MRC-5 cells treated with MK2206 concentration gradient. G Western blotting detection of LC3-II, P62, Collegen I protein expression in MRC-5 cells treated with MK2206. H EDU detection of cell proliferation after MK2206 treatment of MRC-5 cells. I CO-IP detection of AKT binding to TMEM175 protein. J Lyso-Tracker Red staining detects lysosomal changes after co-treatment of SC79 with interfering TMEM175 in MRC-5 cells. *P < 0.05; **P < 0.01; ***P < 0.001

References

    1. Scherer HU, Häupl T, Burmester GR. The etiology of rheumatoid arthritis. J Autoimmun. 2020;110:102400. doi: 10.1016/j.jaut.2019.102400. - DOI - PubMed
    1. Gravallese EM, Firestein GS. Rheumatoid arthritis—common origins, divergent mechanisms. N Engl J Med. 2023;388(6):529–542. doi: 10.1056/NEJMra2103726. - DOI - PubMed
    1. Graudal N, Nielsen CT, Lindhardsen J. Pirfenidone in rheumatoid arthritis-associated interstitial lung disease. Lancet Respir Med. 2023;11(6):e51. doi: 10.1016/S2213-2600(23)00130-3. - DOI - PubMed
    1. McDermott GC, Doyle TJ, Sparks JA. Interstitial lung disease throughout the rheumatoid arthritis disease course. Curr Opin Rheumatol. 2021;33(3):284–291. doi: 10.1097/BOR.0000000000000787. - DOI - PMC - PubMed
    1. Kadura S, Raghu G. Rheumatoid arthritis-interstitial lung disease: manifestations and current concepts in pathogenesis and management. Eur Respir Rev. 2021;30(160):210011. doi: 10.1183/16000617.0011-2021. - DOI - PMC - PubMed

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