1. To explore the regulatory effect of hypoxic microenvironment on glycogen metabolism and inflammatory phenotype of RA-FLS, and clarify the key role of TNF-α in this process.

2. To reveal the role of TNF-α-induced glycophagy dysfunction in glycogen accumulation and invasive phenotype formation of RA-FLS.

3. To clarify the regulatory mechanism of AMPK/mTORC1 signaling pathway on glycophagy abnormality in RA-FLS.

4. To verify the effect of targeting TNF-α/mTORC1 axis on synovial glycophagy function and its therapeutic effect in collagen-induced arthritis (CIA) mice in vivo.

1. Research methods of At the tissue level, PAS staining and amylase digestion experiments were used to compare the glycogen content in synovial tissues of OA and RA patients, so as to clarify the specific accumulation of glycogen in RA tissues; at the cellular level, FLS were isolated from OA and RA patients, the glycogen content and the expression of HIF-1α in RA-FLS were detected, and the effect of hypoxia on abnormal glycogen accumulation in RA-FLS was observed through hypoxia induction experiments at different time points (0h, 12h, 24h); different concentrations of TNF-α (0, 10, 100 ng/mL) were used to intervene RA-FLS, and their effects on HIF-1α level and glycogen content were observed; through hypoxia induction experiments (0h, 6h, 12h, 24h), qPCR was used to detect the expression of inflammatory factors TNF-α and IL-6 in RA-FLS.

2. Research methods of 100 ng/mL TNF-α was used to intervene RA-FLS for 24h, then glycophagy function was detected; qPCR, Western blot and immunofluorescence were used to detect the expression and co-localization of STBD1 and GABARAPL1; STBD1 and GABARAPL1 were knocked down respectively, and the changes of protein levels were detected; immunofluorescence co-localization was used to observe the co-localization level of STBD1 and LAMP1 to evaluate glycophagy activity; in the functional rescue experiment, GABARAPL1 was overexpressed, cell proliferation was detected by EDU assay, cell apoptosis was detected by flow cytometry, cell migration and invasion were detected by Transwell assay, and the mRNA expressions of MMP1, MMP3, CCL2, IL-6, OPG and RANKL were detected by qPCR.

3. Research methods of 10 ng/mL TNF-α was used to intervene RA-FLS for 24 hours, then glycolysis detection was performed; qPCR was used to detect the mRNA expressions of key glycolytic enzymes such as HK2, LDHA, PFK and PGK1, and lactate detection kit was used to determine the intracellular lactate content; exogenous sodium lactate (10nM, 24h) treatment and LDHA inhibitor (FX11) rescue experiment were carried out; Western blot was used to detect the expressions of p-AMPK and p-mTOR; rapamycin (mTORC1 inhibitor) was used to treat cells, and immunofluorescence staining and nucleocytoplasmic separation experiment were used to observe TFEB localization and explore its effect on TFEB nuclear translocation.

4. Research methods of CIA mouse model was established and divided into normal control group, CIA model group, CIA + Etanercept (TNF-α inhibitor) group, CIA + Rapamycin (mTORC1 inhibitor) group and combined intervention group. Intraperitoneal injection was used for administration, and arthritis score was monitored every two days; HE staining of joint tissues was used to evaluate synovial hyperplasia, inflammatory infiltration and bone destruction; immunohistochemistry was used to detect the expressions of key glycophagy proteins STBD1 and GABARAPL1 in synovial tissues; ELISA was used to detect serum IL-6 level, and qPCR was used to detect the mRNA expressions of HIF-1α, TNF-α and IL-17 in synovial tissues.

1. Research methods of At the tissue level, PAS staining and amylase digestion experiments were used to compare the glycogen content in synovial tissues of OA and RA patients, so as to clarify the specific accumulation of glycogen in RA tissues; at the cellular level, FLS were isolated from OA and RA patients, the glycogen content and the expression of HIF-1α in RA-FLS were detected, and the effect of hypoxia on abnormal glycogen accumulation in RA-FLS was observed through hypoxia induction experiments at different time points (0h, 12h, 24h); different concentrations of TNF-α (0, 10, 100 ng/mL) were used to intervene RA-FLS, and their effects on HIF-1α level and glycogen content were observed; through hypoxia induction experiments (0h, 6h, 12h, 24h), qPCR was used to detect the expression of inflammatory factors TNF-α and IL-6 in RA-FLS.

2. Research methods of 100 ng/mL TNF-α was used to intervene RA-FLS for 24h, then glycophagy function was detected; qPCR, Western blot and immunofluorescence were used to detect the expression and co-localization of STBD1 and GABARAPL1; STBD1 and GABARAPL1 were knocked down respectively, and the changes of protein levels were detected; immunofluorescence co-localization was used to observe the co-localization level of STBD1 and LAMP1 to evaluate glycophagy activity; in the functional rescue experiment, GABARAPL1 was overexpressed, cell proliferation was detected by EDU assay, cell apoptosis was detected by flow cytometry, cell migration and invasion were detected by Transwell assay, and the mRNA expressions of MMP1, MMP3, CCL2, IL-6, OPG and RANKL were detected by qPCR.

3. Research methods of 10 ng/mL TNF-α was used to intervene RA-FLS for 24 hours, then glycolysis detection was performed; qPCR was used to detect the mRNA expressions of key glycolytic enzymes such as HK2, LDHA, PFK and PGK1, and lactate detection kit was used to determine the intracellular lactate content; exogenous sodium lactate (10nM, 24h) treatment and LDHA inhibitor (FX11) rescue experiment were carried out; Western blot was used to detect the expressions of p-AMPK and p-mTOR; rapamycin (mTORC1 inhibitor) was used to treat cells, and immunofluorescence staining and nucleocytoplasmic separation experiment were used to observe TFEB localization and explore its effect on TFEB nuclear translocation.

4. Research methods of CIA mouse model was established and divided into normal control group, CIA model group, CIA + Etanercept (TNF-α inhibitor) group, CIA + Rapamycin (mTORC1 inhibitor) group and combined intervention group. Intraperitoneal injection was used for administration, and arthritis score was monitored every two days; HE staining of joint tissues was used to evaluate synovial hyperplasia, inflammatory infiltration and bone destruction; immunohistochemistry was used to detect the expressions of key glycophagy proteins STBD1 and GABARAPL1 in synovial tissues; ELISA was used to detect serum IL-6 level, and qPCR was used to detect the mRNA expressions of HIF-1α, TNF-α and IL-17 in synovial tissues.

1. Conclusion of RA-FLS have the characteristics of hypoxic activation, and hypoxia can induce a large amount of intracellular glycogen accumulation; TNF-α plays a key role in the regulation of RA-FLS glycogen metabolism and inflammatory phenotype by hypoxia; hypoxia can up-regulate TNF-α secretion, and TNF-α can further aggravate glycogen accumulation.

2. Conclusion of TNF-α can induce glycophagy dysfunction in RA-FLS, thereby mediating intracellular glycogen accumulation and the formation of invasive phenotype, while GABARAPL1 can reverse the above abnormal phenotypes and play a negative regulatory role.

3. Conclusion of TNF-α can induce glycolytic metabolic reprogramming in RA-FLS, inhibit AMPK pathway and activate mTORC1 pathway, thereby inhibiting TFEB nuclear translocation, reducing GABARAPL1 transcription level, and ultimately leading to glycophagy abnormality in RA-FLS; lactate, as a glycolytic product, is involved in the regulation of AMPK/mTORC1 pathway mediated by TNF-α.

4. Conclusion of Glycophagy dysfunction is a key pathological feature of synovium in CIA mice; targeting TNF-α/mTORC1 axis can effectively rescue synovial glycophagy function and improve pathological damage of arthritis in CIA mice, providing a new target and experimental basis for the treatment of rheumatoid arthritis.

5. Overall research conclusion: Hypoxic conditions can induce RA-FLS to secrete a large amount of TNF-α, thereby significantly enhancing glycolytic metabolism; this metabolic reprogramming blocks the nuclear translocation of autophagy/lysosome master transcription factor TFEB by inhibiting AMPK and activating mTORC1; the inactivation of TFEB leads to the down-regulation of its downstream target gene GABARAPL1, thereby impairing the STBD1-mediated glycogen autophagy process. This study first reveals a new mechanism by which hypoxia inhibits glycogen autophagy through the TNF-α/mTORC1/TFEB axis, thereby promoting RA-FLS activation.