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

Eight-week-old male C57BL/6 mice with a body weight ranging from 20 to 25 g were randomly assigned to one of three groups: (I) Ctrl (Control), (II) CIA (collagen-induced arthritis models), and (III) CIA + Res (CIA models treated with resveratrol at 10 mg/kg). Male C57BL/6 mice were procured from Jiangsu Ji Cui Biotechnology Co., Ltd. (Nanjing, China) and acclimated to laboratory conditions at Anhui Medical University for a minimum of 1 week before the experiment. The induction of CIA in mice involved the use of complete Freund’s adjuvant (CFA) and chicken type II collagen (CCII). CCII (Chondrex, Woodinville, WA, USA) at a concentration of 2 g/L was dissolved in 0.05 mol/L glacial acetic acid, thoroughly stirred, and stored at 4 °C overnight. Subsequently, CFA (Chondrex, Woodinville, WA, USA) was mixed with an equivalent volume of the dissolved Col II and completely emulsified in an ice bath using a homogenizer. For the CIA + Res group, animals received resveratrol (Aladdin, Shanghai, China) via oral gavage, dissolved in 0.5% sodium carboxymethylcellulose (carrier) at a final concentration of 1 mg/mL, with a daily dosage of 0.1 mL per 10 g of body weight, administered for 10 days. The control group received an equivalent volume of normal saline via tracheal and tail root injection, serving as the negative control. The resveratrol dosage was determined based on prior research findings [30]. All animals were euthanized 24 h after the final resveratrol treatment. Lung tissue was excised, with a section reserved for pathological analysis. The remaining lung tissue was promptly frozen in liquid nitrogen to facilitate subsequent bioanalysis.

Following fixation with 4% neutral paraformaldehyde, knee tissue specimens underwent a 2-week immersion in EDTA decalcification solution (Boster, Wuhan, China) under room temperature agitation. Subsequently, they were subjected to standard dehydration and paraffin embedding procedures. Paraffin sections were approximately 3.5 µm thick, and these sections were deparaffinized and subjected to a series of alcohol and distilled water washes. Hematoxylin staining was performed for 15 s, followed by a 20-min tap water rinse to counterstain. Subsequently, 5% eosin staining for 3 min was carried out, and the sections were rapidly dehydrated. Finally, a neutral resin was used to seal the slides. Lung tissue specimens followed a similar process, including fixation, dehydration, paraffin embedding, and sectioning at a thickness of approximately 3.5 µm. HE staining was performed on these lung tissue sections as well.

Masson staining (Solarbio, Beijing, China) was employed to identify fibrosis. The cells were subjected to iron hematoxylin staining for 8 min at room temperature for nuclear staining, followed by differentiation to achieve a blue color. Subsequently, they were stained with aniline blue for 1.5 min, washed with weak acid, dehydrated, and sealed. Finally, observations were made under a microscope (Nikon Eclipse 80I), and analysis was conducted using Image Pro 6 (Media Contronetics Inc., Bethesda, MD, USA).

Following the standard dewaxing of paraffin sections, endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ for 20 min. Subsequently, antigen retrieval was performed using a microwave for 10 min, and the primary antibody was incubated at 4 °C overnight. The addition of the secondary antibody was followed by a 60-min incubation at room temperature. DAB (Solarbio, Beijing, China) was utilized for color development, and the slides were counterstained with hematoxylin before sealing with neutral resin. Five randomly selected areas within each section were examined under a light microscope. Positive expression was indicated by yellow–brown staining in the cytoplasm or cytoplasmic membrane. The proportion of positive area (positive area/total area) was quantified using ImageJ software.

Following the kit instructions (Solarbio, Beijing, China), approximately 0.2 g of the sample was weighed into a glass tube. The tissue was minced as finely as possible for digestion, and 2 mL of extraction solution was added before bringing it to a boil. After cooling, the pH was adjusted to the range of 6–8 using 10 mol/L NaOH (approximately 1 mL), and then distilled water was added to reach a final volume of 4 mL. Subsequently, centrifugation was carried out at 16,000 rpm and 25 °C for 20 min. The spectrophotometer wavelength was set to 560 nm, and the supernatant was collected for measurement.

Three-dimensional structural representations of the crucial active components of resveratrol were acquired from the PubChem database to serve as ligand files for subsequent molecular docking. The 3D structures of the target proteins were retrieved as receptor files from the RCSB database (https://​www.​pdbus.​org/​). AutoDock (version 1.5.6) was employed to extract small ligand molecules from the receptors, perform side chain corrections, and introduce hydrogen atoms. Subsequently, AutoDock Vina (version 1.1.2) was utilized for molecular docking. A protein binding affinity below − 5 kcal/mol indicates significant binding activity with the compound. The docking outcomes for the compounds and proteins displaying the most favorable conformations were subjected to analysis and visualization using PyMol software.

Gromacs was utilized to conduct molecular dynamics simulations on the resveratrol-TGF-β1 protein complex, which exhibited the highest binding free energy in molecular docking. Protein modeling employed aber14SB force field parameters, while small molecule ligands were modeled with Gaff generic force field parameters. The OPC water model was employed to introduce solvents into the protein–ligand system. Subsequently, a water tank was established and supplemented with Na⁺ and Cl⁻ to achieve system equilibrium. Energy optimization was carried out employing the most rapid descent and conjugate gradient methods. Conformational constraints were applied, followed by Nvt pre-equilibrium and Npt pre-equilibrium, culminating in MD simulations. Binding free energy serves as the primary criterion for assessing drug molecule activity, with lower values indicating greater complex stability.

The human embryonic lung fibroblast cell line (MRC-5) was procured from the American Typical Collection of Biological Resources (ATCC, Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies/Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics (containing 100 U/mL penicillin, 100 μg/mL streptomycin, Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained in a 37 °C incubator with 5% CO₂. To induce a cellular model of RA-ILD, recombinant TGF-β1 and IL-1β were added to the cell culture medium for a 24-h incubation period. TGF-β1, IL-1β, SC79, MK2206, chloroquine (CQ), and bafilomycin A1 (BAFA1) were procured from MedChemExpress, while MG132 was obtained from Selleckchem.

SiRNA targeting TMEM175 (Si-TMEM175) was custom-designed and synthesized by General Biologicals. Cells were transfected with SiRNA at a concentration of 50 nmol/L using Lipo2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s guidelines. Subsequently, the transfected cells were utilized for experiments. The sequence of Si-TMEM175 is provided in Table 1.

MRC-5 cells were seeded in 96-well plates at a density of 3 × 10³ cells per well. After 6–12 h, the cells were stimulated with IL-1β and TGF-β1 for 24 h. Subsequently, following a 24-h treatment with resveratrol, the cells were further incubated for 1–4 h with the addition of 10 μL of CCK-8 solution (Biosharp, Beijing, China) to each well. The absorbance of the cells at 450 nm was assessed using an enzyme-linked marker to evaluate the impact of resveratrol on cell viability.

Culture an appropriate cell number in a 6-well plate. Once the cell culture has returned to its normal state overnight, proceed with the desired treatments. Prepare the EdU working solution (Beyotime Biotechnology, Shanghai, China) and add the preheated EdU working solution at 37 °C to the 6-well plate, incubating the cells for 2 h. Fix the Edu-labeled cells by adding 1 mL of fixative and incubating for 15 min at room temperature. Subsequently, incubate each well with 1 mL of permeabilizing solution for 10–15 min at room temperature. Add 0.5 mL of Click reaction solution to each well and incubate for 30 min at room temperature, away from light. Stain the cell nuclei with Hoechst 33342, followed by fluorescence detection.

ROS levels were assessed using the DCFH-DA green fluorescent probe (Beyotime Biotechnology, Shanghai, China). DCFH-DA was diluted 1000-fold with DMEM to create a working solution. Subsequently, 1–2 mL of the working solution was added to completely cover the cells growing on the culture dish and incubated at 37 °C for 20 min in the absence of light. Afterward, the cells were washed three times with PBS buffer and imaged using an inverted fluorescence microscope. The alterations in ROS levels in MRC-5 cells were analyzed using ImageJ software.

The mitochondrial membrane potential was assessed using the mitochondrial membrane potential kit from Beyotime Biotechnology (Shanghai, China). Cells were seeded and incubated overnight at 37 °C. Following the instructions provided with the JC-1 reagent kit, JC-1 was diluted 1000-fold to prepare the working solution. Subsequently, 1–2 mL of the working solution was added to fully cover the cells on the culture dish and incubated at 37 °C in the absence of light for 15 min. The cells were then washed three times with PBS buffer. Images were captured using an inverted fluorescence microscope, and changes in the membrane potential of MRC-5 cells were analyzed using ImageJ software.

Cells were transiently transfected with the mCherry-GFP-LC3 construct using EZ Cell Transfection Reagent (Shanghai Life-iLab Biotech, AC04L091). Transfection was performed by adding 3 μg of mCherry-GFP-LC3 plasmid to 9 μL of the transfection reagent, and the culture medium was refreshed every 24 h. After 48 h of cell model treatment, the number of mCherry and GFP fluorescent puncta was directly observed using a laser confocal microscope (Leica SP8, Wetzlar, Germany).

The cells to be examined were subjected to three washes with PBS. Subsequently, 1 mL of MDC staining solution (Beyotime Biotechnology, Shanghai, China) was added to each well, and the cells were incubated in a cell culture incubator at 37 °C for 30 min while protected from light. Afterward, the cells were washed three times with Assay Buffer, with each wash involving 0.8–1 mL of Assay Buffer. Finally, the Assay Buffer was aspirated, and 1 mL of fresh Assay Buffer was added. The green fluorescence was observed using a fluorescence microscope with UV excitation light.

A small quantity of Lyso-Tracker Red (Beyotime Biotechnology, Shanghai, China) was added to the cell culture medium at a ratio of 1:20,000, resulting in a final concentration of 50 nM. The cell culture medium was then aspirated, and the prepared working solution was added, followed by incubation at 37 °C for 30 min. Subsequently, the Lyso-Tracker Red stained working solution was removed, and a fresh cell culture medium was added. Photographic observation was carried out using a laser confocal microscope.

Total protein was extracted from mouse lung tissues and cells using a prepared SDS lysis buffer for protein blotting analysis. The proteins were separated on SDS–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). The membrane was then blocked with 5% skimmed milk for 2 h. Antibodies against β-actin (Abcam, #ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), TGF-β1 (ImmunoWay, #YT4632), P-NFKB (Wanleibio, #WL02169), NFKB (Bioss, #bsm-33117M), TNF-α (Proteintech, #60291-1-Ig), IL-1β (Wanleibio, #WLH3903), HIF-1a (Proteintech, #20960-1-AP), SOD1 (Wanleibio, #WL01846), SOD2 (Boster, #BA4566), LC3-II (Cell Signaling Technology, #12741), P62 (Cell Signaling Technology, #16177S), LAMP2 (Proteintech, #66301-1-Ig), P-mTOR (Abmart, #T56571), mTOR (Abmart, #T55306), P-ULK1 (ImmunoWay, #YP1544), ULK1 (Abmart, #T56902), Beclin1 (Boster, #PB0014), Atg5 (Bioss, #bs-4005r), BNIP3 (Santa Cruz Biotechnology, #sc-56167), P-AKT (Abmart, #T40067), AKT (Abmart, #BM4400), and TMEM175 (Proteintech, #19925-1-AP) were used. The PVDF membrane was incubated with these antibodies at 4 °C overnight. Subsequently, an HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, followed by detection using the ECL luminescence system.

Transfer 25–50 μL of protein A/G magnetic beads (GenScrip, New Jersey, USA) into 1.5 mL tubes and add 400 μL of Wash Buffer (PBST) to the beads. Dilute Antibody TMEM175 with Wash Buffer to achieve a final concentration of 5–50 μg/mL. Combine 400 μL of the diluted Ab with the Protein A/G beads and incubate at 2 °C for 4 h with rotation. Wash the beads by adding 400 μL of Wash Buffer and gently agitating. Place the tube in a magnetic holder, collect the beads on the side of the tube, discard the supernatant, and repeat this step 4 times. Remove the tube from the magnetic separator, add the sample containing the antigen (Ag), and gently pipette to resuspend the protein A/G bead-Ab complex. Incubate at 2 °C for 4 h to allow Ag to bind to the Protein A/G Bead-Ab complex. Add 400 μL of Wash Buffer to the tube, spin for 5 min, perform a 1-min magnetic separation, and remove the supernatant. Finally, add 25–50 μL of elution buffer to the tube containing the magnetic bead-Ab-Ag complex and spin for 5 min to elute the protein complex.

MRC-5 cells were plated in 48-well plates at a density of 1 × 10⁴ cells per well. After 48 h of cell treatment, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then rinsed with PBS. Subsequently, the cells were treated with 0.1% Triton-X for 20 min and rinsed again. Afterward, they were blocked with 5% bovine serum albumin (BSA, Solarbio, Beijing, China) for 60 min at room temperature, followed by incubation with antibodies (diluted 1:200) such as LC3-II, P62, etc., overnight at 4 °C. The cells were then rinsed and incubated for 1 h in the dark with secondary antibodies (goat anti-mouse antibody, Proteintech or goat anti-rabbit antibody, Proteintech). Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added to the cells for a 10-min incubation in the dark. Finally, the samples were sealed with an anti-fading mounting medium (Beyotime Biotechnology, Shanghai, China) and observed using an inverted fluorescence microscope.

Total RNA was isolated from mouse lung tissue using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). TRIzol was added to the lung tissue powder and allowed to stand at room temperature for 5 min. Subsequently, 200 μL of chloroform was added to each sample and vortexed. After a 5-min incubation, the samples were centrifuged at 12,000 rpm and 4 °C for 15 min. The upper aqueous phase was transferred, and 400 μL of isopropanol was added, followed by a 10-min incubation and centrifugation at 12,000 rpm and 4 °C for 10 min to collect the RNA pellet. The RNA pellet was washed with 70% ethanol, centrifuged, air-dried, and then resuspended in DEPC water. Finally, RNA concentration and purity were determined using UV spectrophotometry. Reverse transcription of total RNA was performed according to the instructions of the cDNA Reverse Transcription Kit. Quantitative PCR was carried out using SYBR Green (Beyotime Biotechnology, Shanghai, China) on a Roche LightCycler 480 real-time fluorescent quantitative PCR system. After completion of the reaction, the cycle threshold (Ct) values were determined, and the relative RNA levels of each sample were calculated using the 2^−ΔΔCt method, with normalization to β-actin levels. Primer sequences are provided in Table 1.

Cell culture and treatment followed the same protocol as described above. Single-cell suspensions were prepared and fixed in 70% ethanol by volume. RNase A was added, followed by a 30-min incubation in a 37 °C water bath. Then, PI (Bestbio, Shanghai, China) staining was added, and mixed, and the samples were kept in the dark at 4 °C for 30 min. Flow cytometry (BD Celesta, USA) was conducted to record red fluorescence with an excitation wavelength of 488 nm.

Earlier studies have reported the effectiveness of resveratrol in mitigating disease progression in rheumatoid arthritis [10]. Nevertheless, the potential effect of resveratrol in RA-associated interstitial lung disease (RA-ILD) is seldom addressed. In this study, we established a mouse model of RA-ILD treated with resveratrol. Hematoxylin and eosin (HE) staining of mouse joints revealed that resveratrol mitigated inflammation and damage in these joints (Fig. 1A). Both HE staining and Masson staining of lung tissue demonstrated the effective attenuation of fibrosis in RA-ILD by resveratrol (Fig. 1B, C). qRT-PCR results indicated a significant inhibition by resveratrol in the mRNA expression of Collagen I, Collagen III, and FN1 (Fig. 1D). Hydroxyproline assay further demonstrated the attenuation of fibrosis in RA-ILD by resveratrol (Fig. 1E). Additionally, immunohistochemistry and Western blot results illustrated that resveratrol not only decreased Collagen I expression but also markedly inhibited TGF-β1 expression (Fig. 1F–H).

To assess the potential of resveratrol in mitigating the progression of RA-ILD through the inhibition of TGF-β1 production, we developed a molecular docking model of resveratrol targeting TGF-β1. The binding free energy of the model complex was − 5.8 kcal/mol, indicating a relatively stable binding affinity between resveratrol and TGF-β1 protein (Fig. 2A). Molecular dynamics simulation confirmed the stability of the binding between resveratrol and TGF-β1 protein. The equilibrium of the simulation system was evaluated using RMSD, revealing that the protein complex started to stabilize at 50 ns. RMSF analysis reflected the freedom degree of each residue within the molecule. These findings collectively suggest that resveratrol exerts its pharmacological effects through direct binding to the TGF-β1 protein (Fig. 2B). Subsequently, we induced MRC-5 cells with TGF-β1 + IL-1β (10 ng/mL + 5 ng/mL) to establish an in vitro model. Through Western blot and immunofluorescence experiments, we identified the optimal concentration of resveratrol (10 µm) and evaluated its effect on TGF-β1 protein expression in the cell model. The results indicated a significant inhibition by resveratrol in Collagen I and TGF-β1 protein expression in MRC-5 cells (Fig. 2C–E). Furthermore, CCK8 and EDU assays demonstrated that resveratrol attenuated TGF-β1-induced cell proliferation (Fig. 2F, G). The aforementioned findings suggest that resveratrol alleviates fibrosis in RA-ILD by targeting and inhibiting the expression of TGF-β1.

To assess whether resveratrol mitigated inflammation and oxidative stress in RA-ILD, we analyzed the expression of inflammation-related proteins (e.g., TNF-α and IL-1β) using immunohistochemistry, Western blotting, and qRT-PCR. The findings demonstrated that resveratrol reduced inflammation in RA-ILD (Fig. 3A–C). Furthermore, the results from MDA and Western blotting experiments confirmed the reduction in oxidative stress in RA-ILD due to resveratrol treatment (Fig. 3D, E). To further corroborate the alleviation of inflammation and oxidative stress by resveratrol in RA-ILD, we assessed the expression of inflammation and oxidative stress-related proteins in an in vitro cellular model. The results were consistent with the in vivo animal experiments, confirming that resveratrol reduced inflammation and oxidative stress in MRC-5 cells (Fig. 3F, G). Moreover, ROS and JC-1 assays revealed that resveratrol decreased the levels of ROS and prevented the decline in cell membrane potential in MRC-5 cells (Fig. 3H). In summary, these findings indicate that resveratrol alleviated inflammation and oxidative stress in RA-ILD.

The study revealed a notable alteration in autophagy levels in RA-ILD, as depicted in the figure below. Immunohistochemistry and Western blotting demonstrated substantial accumulation of P62 in RA-ILD, along with increased expression of LC3-II and LAMP2. However, resveratrol treatment resulted in reduced levels of P62 and LC3-II, with a slight increase in LAMP2 expression (Fig. 4A, B). Furthermore, resveratrol facilitated mTOR activation, inhibited ULK1 phosphorylation (Fig. 4C), and reinstated the expression of Beclin1, Atg5, and Bnip3, which exhibited elevated levels in RA-ILD (Fig. 4D). To further confirm the modulation of autophagy in RA-ILD by resveratrol, we assessed the presence of autophagic vesicles in cell models using MDC (Fig. 4E) and analyzed the protein expression of P62, LC3-II, and LAMP2 through Western blotting and immunofluorescence (Fig. 4F, G). The findings indicated that resveratrol reinstated the disrupted autophagic flux in RA-ILD.

To further elucidate the role of resveratrol in autophagy in RA-ILD, we assessed the expression of autophagy-related RNAs, including TFEB, RAB7A, VAMP8, and VATPASE, using qRT-PCR. The results revealed elevated expression of pro-autophagy lysosomal fusion RNAs in RA-ILD, which was further augmented upon resveratrol treatment (Fig. 5A–D). Subsequently, MRC-5 cells were treated with chloroquine (CQ), bafilomycin A1 (BAFA1), and MG132, and we determined the optimal concentrations (CQ: 20 µm, BAFA1: 40 nm, MG132: 10 µm) based on autophagy protein levels (Fig. 5E–G). We assessed the impact of these three drugs on the expression of autophagy proteins and Collagen I in the cellular model following resveratrol treatment using Western blotting. The results indicated that CQ significantly impeded the effects of resveratrol when compared to the other two drugs, resulting in autophagy and fibrosis levels akin to those observed in RA-ILD (Fig. 5H). To further confirm these findings, we transfected the mCherry-GFP-LC3 plasmid into MRC-5 cells and assessed the progress of autophagy and lysosomal fusion based on alterations in fluorescence (Fig. 5I). In addition, to determine whether the apparent upregulation of LC3-II was due to inhibition of autophagic flux rather than initiation of normal autophagy. We performed experiments using CQ plus TGF-β1 + IL-1β, and the results showed that compared to the CQ group, the LC3-II protein in the CQ + TGF-β1 + IL-1β group was not significantly increased or even slightly decreased, and the P62 protein was not significantly altered. Thus indicating that the upregulation of LC3-II was not due to TGF-β1 + IL-1β initiating normal cellular autophagy (Fig. 5J).

To further investigate how resveratrol regulates the fusion of autophagic lysosomes, we found that TMEM175 was highly expressed in RA-ILD using qRT-PCR and showed a decrease in the resveratrol-treated group (Fig. 6A). Western blotting with immunohistochemistry confirmed this result (Fig. 6B, C). Next, we re-evaluated TMEM175 expression in an in vitro cellular model, which showed consistent results with the previous findings (Fig. 6D, E). To delve deeper into the mechanism through which TMEM175 regulates autophagic lysosomal fusion, we employed SiRNA interference. Western blotting was employed to assess the impact of interference (Fig. 6F). Analysis of cellular autophagy following interference with TMEM175 using mCherry-GFP-LC3 labeling demonstrated the restoration of autophagic lysosomal fusion in MRC-5 cells (Fig. 6G). Furthermore, Lyso-Tracker Red staining revealed a decrease in acidic lysosomes within the TGF-β1-induced MRC-5 cell model, and this effect was counteracted by TMEM175 interference (Fig. 6H). Conversely, Western blotting results demonstrated that interference with TMEM175 restored autophagy and suppressed fibrosis in MRC-5 cells (Fig. 6I). The outcomes of EDU staining and flow cytometry assays indicated that interference with TMEM175 suppressed TGF-β1-induced cell proliferation (Fig. 6J, K). These findings imply that resveratrol mitigates fibrosis in RA-ILD by suppressing TMEM175 protein expression, restoring lysosomal pH, and enhancing autophagic lysosomal fusion.

Prior studies have reported the formation of a lysosomal K⁺ channel complex when AKT binds with TMEM175. This complex is activated by growth factors and plays a role in regulating lysosomal K⁺ and H⁺ transport. This regulation occurs via an adaptable effector gate that influences lysosomal pH, thereby impacting autophagic lysosomal fusion [27, 28, 31, 32]. Consequently, we conducted immunohistochemistry and Western blotting on animal models to assess AKT expression levels. The results indicated elevated levels of both P-AKT and AKT in RA-ILD, which decreased following resveratrol treatment (Fig. 7A, B). We subsequently validated these findings through in vitro experiments, wherein we observed elevated AKT expression in TGF-β1-induced MRC-5 cells. Resveratrol treatment effectively reversed the AKT elevation (Fig. 7C, D). To further explore the role of AKT in RA-ILD, we treated MRC-5 cells with an AKT activator (SC79) and an AKT-specific inhibitor (MK2206). We determined the optimal drug concentrations (SC79: 2 μM, MK2206: 4 μM) through Western blotting analysis (Fig. 7E, F). Western blotting results indicated that inhibiting AKT metabolic activation reinstated TGFβ1-induced autophagy and decreased Collagen I expression in MRC-5 cells (Fig. 7G). Additionally, EDU assays demonstrated that inhibiting AKT metabolic activation reversed the proliferation of MRC-5 cells (Fig. 7H). Subsequently, we conducted co-immunoprecipitation experiments to illustrate the formation of a complex between AKT and TMEM175. This complex plays a role in regulating autophagic lysosomal fusion (Fig. 7I). Additionally, Lyso-Tracker Red staining revealed that AKT activation facilitated the formation of the AKT/MEM175 complex and led to a reduction in the number of acidic lysosomes. However, this effect was reversed upon disruption of TMEM175 (Fig. 7J). To summarize, AKT serves as a gating control, facilitating the opening of the TMEM175 ion channel, and consequently, it regulates lysosomal H⁺ transport. However, it alone cannot independently influence lysosomal pH changes.