Strategy for treating MAFLD: Electroacupuncture alleviates hepatic steatosis and fibrosis by enhancing AMPK mediated glycolipid metabolism and autophagy in T2DM rats

Strategy for treating MAFLD: Electroacupuncture alleviates hepatic steatosis and fibrosis by enhancing AMPK mediated glycolipid metabolism and autophagy in T2DM rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Strategy for treating MAFLD: Electroacupuncture alleviates hepatic steatosis and fibrosis by enhancing AMPK mediated glycolipid metabolism and autophagy in T2DM rats Haoru DUAN, Shanshan Song, Rui Li, Suqin Hu, Shuting Zhuang, Shaoyang liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4475748/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Diabetology & Metabolic Syndrome → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Recent studies havehighlighted type 2 diabetes (T2DM) as a significant risk factor for the development of metabolic dysfunction-associated fatty liver disease (MAFLD). This investigation aimed to assess electroacupuncture's (EA) impact on liver morphology and function in T2DM rats, furnishing experimental substantiation for its potential to stall MAFLD progression in T2DM. Methods: T2DM rats were induced by a high-fat diet and a single intraperitoneal injection of streptozotocin, and then randomly assigned to five groups: the T2DM group, the electroacupuncture group, the metformin group, combination group of electroacupuncture and metformin, combination group of electroacupuncture and Compound C. The control group received a standard diet alongside intraperitoneal citric acid - sodium citrate solution injections. After a 6-week intervention, the effects of each group on fasting blood glucose, lipids, liver function, morphology, lipid droplet infiltration, and fibrosis were evaluated. Techniques including Western blotting, qPCR, immunohistochemistry, and immunofluorescence were employed to gauge the expression of key molecules in AMPK-associated glycolipid metabolism, insulin signaling, autophagy, and fibrosis pathways. Additionally, transmission electron microscopy facilitated the observation of liver autophagy, lipid droplets, and fibrosis. Results: Our studies indicated that hyperglycemia, hyperlipidemia and IR promoted lipid accumulation, pathological and functional damage, and resulting in hepatic steatosis and fibrosis. Meanwhile, EA enhanced the activation of AMPK, which in turn improved glycolipid metabolism and autophagy through promoting the expression of PPARα/CPT1A and AMPK/mTOR pathway, inhibiting the expression of SREBP1c, PGC-1α/PCK2 and TGFβ1/Smad2/3 signaling pathway, ultimately exerting its effect on ameliorating hepatic steatosis and fibrosis in T2DM rats. The above effects of EA were consistent with metformin. The combination of EA and metformin had significant advantages in increasing hepatic AMPK expression, improving liver morphology, lipid droplet infiltration, fibrosis, and reducing serum ALT levels. In addition, the ameliorating effects of EA on the progression of MAFLD in T2DM rats were partly disrupted by Compound C, an inhibitor of AMPK. Conclusions: EA upregulated hepatic AMPK expression, curtailing gluconeogenesis and lipogenesis while boosting fatty acid oxidation and autophagy levels. Consequently, it mitigated blood glucose, lipids, and insulin resistance in T2DM rats, thus impeding liver steatosis and fibrosis progression and retarding MAFLD advancement. T2DM MAFLD EA AMPK signaling pathway autophagy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Non-Alcoholic Fatty Liver Disease (NAFLD), recently redefined as Metabolic Associated Fatty Liver Disease (MAFLD), presents a significant health burden worldwide, particularly among individuals with Type 2 Diabetes Mellitus (T2DM). The progression of MAFLD from simple hepatic steatosis to steatohepatitis and fibrosis poses substantial risks to affected individuals, particularly those with comorbid T2DM ( 1 ). Studies had consistently shown that T2DM patients faced a substantially elevated risk up to 70% − 80% of developing MAFLD compared to non-diabetic counterparts ( 2 – 4 ). Importantly, the convergence of T2DM and MAFLD escalated the risk of cardiovascular complications due to compromised metabolic control ( 5 – 8 ). A recent prospective case-control study involving 2103 T2DM patients underscored this risk, revealing an association between MAFLD and increased cardiovascular disease risk over a five-year follow-up period, even after adjustments for other pertinent risk factors were made ( 9 ). Hence, the evaluation and management of MAFLD, even in its milder forms, are imperative for mitigating cardiovascular risk and improving overall mortality rates among individuals with T2DM. The pathological process of MAFLD is intricately intertwined with insulin resistance (IR), a condition characterized by decreased sensitivity of cells to the effects of insulin hormone. In MAFLD initiation, the primary manifestation is simple steatosis, characterized by the accumulation of triglycerides (TG) in over 5% of hepatocytes ( 10 ) ( 11 ). This accumulation is significantly influenced by IR, which serves as a driving force in the development and progression of MAFLD. One of the key consequences of IR is the increased influx of fatty acids into the liver, which, coupled with elevated hepatic de novo lipogenesis (DNL), results in heightened TG levels within hepatocytes ( 12 – 14 ). Furthermore, inhibition of peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) reduces fatty acid oxidation (FAO), further contributing to TG accumulation ( 10 , 15 ). The failure of insulin to effectively regulate liver glucose uptake and production exacerbates hyperglycemia and IR, thereby exacerbating the pathogenic processes underlying MAFLD ( 16 ). Persistent IR, coupled with increased free fatty acid (FFA) influx, induces mitochondrial dysfunction, reactive oxygen species (ROS) production, and toxic lipid accumulation, which in turn exacerbates hepatic damage ( 17 ). As MAFLD progresses, it encompasses fatty infiltration, lobular inflammation, and can manifest into severe forms such as non-alcoholic steatohepatitis (NASH) ( 18 , 19 ). NASH, if left unchecked, may advance to perisinusoidal fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC) ( 1 , 20 , 21 ). Given the reversible nature of MAFLD in its early stages, timely intervention becomes paramount in altering its trajectory and curbing its escalating prevalence ( 22 , 23 ). Thus, there is an imperative need to address the underlying pathological factors, particularly focusing on interventions aimed at mitigating diabetes-induced liver injury. Autophagy, a cellular process essential for maintaining homeostasis, serves as a protective mechanism against liver injury induced by various metabolic insults ( 24 – 26 ). Specifically, in the context of MAFLD, dysregulated lipid metabolism and oxidative stress overwhelms cellular defenses, leading to hepatocyte dysfunction and the progression of liver pathology. Autophagy in hepatocytes plays a pivotal role in mitigating these effects by removing damaged organelles and lipid droplets, thus preventing excessive cell death and fibrosis ( 24 , 27 ). However, when autophagy is impaired, as seen in MAFLD, the liver becomes more susceptible to injury and fibrotic progression ( 28 – 30 ). Studies have demonstrated that knocking out autophagy-related genes exacerbates hepatic fibrosis, underscoring the protective role of autophagy in liver pathology ( 31 ). To optimize therapeutic outcomes in MAFLD, addressing metabolic disturbance, steatosis, and liver damage is crucial ( 32 ). One promising strategy for the treatment of T2DM-induced MAFLD involves targeting the AMP-activated protein kinase (AMPK) pathway, which plays a crucial role in cellular energy homeostasis and metabolism regulation ( 33 ). Activation of AMPK has been shown to enhance glycolipid metabolism and autophagy, both of which are essential processes for mitigating the adverse effects of insulin resistance in MAFLD. By enhancing glycolipid metabolism, AMPK activation alleviates the excess accumulation of triglycerides in hepatocytes by promoting the conversion of glucose into energy rather than storing it as lipids ( 34 – 37 ). Additionally, autophagy induction facilitated by AMPK activation can aid in the removal of damaged mitochondria and toxic lipid species, thus mitigating the progression of hepatic damage in MAFLD ( 38 , 39 ). Moreover, targeting AMPK signaling also improves insulin sensitivity, thereby addressing one of the central pathological features of MAFLD( 40 ). Therefore, interventions aimed at enhancing AMPK-mediated glycolipid metabolism and autophagy hold promise as potential therapeutic avenues for addressing insulin resistance in the pathological process of MAFLD. While several compounds are undergoing clinical investigation for MAFLD management, none have received specific approval yet ( 41 ). Acupuncture, a unique external therapy in traditional Chinese medicine with a lengthy history of treating T2DM in China, has been reported to be effective against T2DM, IR, and diabetic liver injury ( 42 – 45 ). Recent studies, including our unpublished research, had indicated that EA's hypoglycemic and hypolipidemic effects protected the liver's morphology and function in T2DM rats by reducing inflammation and lipid droplet infiltration in liver tissue ( 46 , 47 ). These findings underscored EA's therapeutic potential in ameliorating T2DM with MAFLD, with the underlying molecular mechanism associated with AMPK activation. Building upon these promising results and “prevent before the disease exacerbats” thought in traditional chinese medicine (TCM), we hypothesized that EA might prevent hepatic steatosis and fibrosis in diabetes-induced hepatic dysfunction by enhancing the AMPK signaling pathway, resulting in the upregulation of glycolipid metabolism and autophagy and inhibiton of TGFβ/SMAD3 signaling pathways. Therefore, our present study primarily investigated EA's potential effect and mechanism of treatment and its specific relationship with hepatoprotection and anti-fibrosis in T2DM rats induced by a high-fat diet (HFD) and injection of STZ, aiming to provide therapeutic strategies for preventing or delaying the progression of MAFLD. Material and Methods Establishment of T2DM animal model Male Wistar rats of Specific Pathogen Free (SPF) grade, weighing 150–180 g, were procured from Sibeifu (Beijing) Biotechnology Co., Ltd. These rats were maintained at 23 ± 2°C, with a humidity of 40% ± 5%, under a 12-hour light/dark cycle. After acclimatization for 7 days, 10 rats were randomly assigned to the Control group (Con), provided with ad libitum access to water and standard rat chow. The remaining rats were induced to develop a T2DM model through a high-fat diet combined with STZ injection. The induction protocol involved feeding the rats a high-fat diet (comprising 67% regular rodent chow, 20% sucrose, 10% lard, 2.5% cholesterol, and 0.5% sodium cholate) for 4 weeks, followed by an overnight fast of 20 hours, and subsequent intraperitoneal injection with a 2% solution of streptozocin (STZ) (dissolved in 0.1 mmol/L citrate buffer solution with a pH of 4.2–4.5) at a dose of 35 mg/kg. The Con group rats were intraperitoneally injected with 0.1 mol/L pH = 4.2 citric acid - sodium citrate buffer. The criterion for successful T2DM model preparation was set as Random Blood Glucose (RBG) ≥ 16.7mmol/L and/or Fasting Blood Glucose (FBG) ≥ 11.1 mmol/L. Throughout the 14-day model evaluation and 6-week interventions, rats, excluding those in the Con group, were fed a high-fat diet. A total of 60 rats were included as model animals, and they were randomly allocated into the following groups based on FBG levels: Control (Con), T2DM, Electroacupuncture (EA), Metformin (Met), Electroacupuncture combined with Metformin (EA + Met), and Electroacupuncture combined with Compound C (EA + compd C), each comprising 10 rats. Rats with EA treatment involved the use of disposable sterile acupuncture needles (0.13*7 mm) at bilateral acupoints, including Pishu (BL 19), Weiwanxiashu (EX B3), Zusanli (ST 36), and Sanyinjiao (SP 6). The needles were inserted to a depth of 4–6 mm. Additionally, Weiwanxiashu (EX B3) and Zusanli (ST 36) were connected to an electroacupuncture instrument set to a continuous wave, with a frequency of 15 Hz and an amplitude of 2–4 mA, for 20 minutes per session, Once a day. In contrast, rats in the Met group received a daily gavage of 60 mg/mL metformin solution, dosed at 5 mL/kg body weight. Meanwhile, rats in the EA + Met group underwent both EA and Met treatment once a day. The EA session lasted for 20 minutes, while Met was administered at a dosage of 300 mg/kg. Another group, the EA + compd C group, was subjected to intraperitoneal injection of Compound C at a dosage of 10 mg/kg, in addition to the daily 20-minute acupuncture session. Rats in the control and T2DM groups underwent immobilization similar to that of the EA group but without any other interventions. All interventions were carried out six times a week over the six-week period. At the end of the six-week intervention, the rats were sacrificed for further analysis. Plasma and liver samples were collected and stored for subsequent examinations. Measurement of FBG, blood lipids, weight, serum insulin (INS), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin (ALB) FBG levels of all rats were measured through tail vein using the Roche Accu-Chek blood glucose meter before modeling, before intervention, and at the end of each week during the intervention period, following a 12-hour fast. The animal weight were measured before modeling, before and after intervention. The serum was separated through centrifugation at 3000 r/min, at 4℃ for 15 min, and serum concentrations of TC, TG, LDL, HDL, AST, ALT and ALB were determined by enzyme colorimetry. Insulin levels were measured by radioimmunoassay. Homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity (HOMA-ISI) were computed with the following formula: HOMA-IR = (Fasting glucose (mmol/L) × Fasting serum insulin (µU/mL))/22.5. HOMA-ISI = 22.5 / {Fasting glucose (mmol/L) × fasting serum insulin (µU/mL)}. Hematoxylin-eosin (HE) staining Hematoxylin and eosin (H&E) staining was performed to examine liver morphology. Livers were collected in 4% paraformaldehyde solution for 24 h, and embedded in paraffin wax blocks after dehydrating through a serial alcohol gradient. All sections were cut at 5 µm sections through a microtome, and processed for staining with H&E. After staining, the sections were dehydrated with ascending concentrations of ethanol and xylene. Images were captured using an optical microscope (BX53; Olympus Optical). Oil Red O staining (G1261) Oil Red O staining was used to evaluate liver steatosis. Mix Oil Red O stain A and B with the radio of 3:2 and place for 10 min to form modified Oil Red O stain solution. 10-µm-thick frozen liver sections were washed by water after fix in 10% formalin for 10 min and soaked in 60% isopropanol for 30s. Staining was performed in modified Oil Red O stain solution for 10min. The tissue slices were placed in 60% isopropanol to continue colour separation. After staining, the sections were staining by Mayer’s Hematoxylin solution for 1 min and rinsed by tap water for 10 min. Images were captured using an optical microscope (BX53; Olympus Optical). Masson staining (G1346) Masson staining was used to evaluate liver fibrosis. 5-µm-thick paraffin sections were routinely dewaxed to distilled water and incubated in Mordant Solution (12h), Celestite Blue Solution (2min), Mayer hematoxylin (2min). After differentiation with Acid Differentiation Solution, the sections were washed with distilled water to stop differentiation, and then treated with Ponceau-Acid Fuchsin Solution (10min), Phosphomolybdic Acid Solution (10min), Aniline Blue Solution (5min). Images were captured using an optical microscope (BX53; Olympus Optical). Immunohistochemical staining The liver paraffin sections underwent dewaxing to water and were subsequently subjected to heating in sodium citrate antigen retrieval solution in a microwave oven for 15 minutes. Following PBS washing, goat serum was applied to block the sections for 10 minutes. A specific primary antibody (listed in Supplementary Table 1) was then applied for overnight incubation at 4 ℃, succeeded by incubation with immunohistochemical secondary antibodies for 10 minutes at room temperature. After a 3-minute incubation with DAB solution, all sections were examined using an optical microscope (Nikon 80i, Japan). Immunofluorescence staining The liver paraffin sections were dewaxed to water and heated in sodium citrate antigen retrieval solution in a microwave oven for 15 minutes. Subsequent to PBS washing, the sections were blocked with immunostaining blocking solution for 1 hour. Overnight incubation with a specific primary antibody (listed in Supplementary Table 1) at 4 ℃ was followed by incubation with fluorescent secondary antibodies for 2 hours at room temperature in the dark. After a 5-minute incubation with DAPI, all sections were observed via fluorescent microscopy (Nikon 80i, Japan). Western blotting Liver tissue was lysed using RIPA lysis buffer containing protein phosphatase inhibitor, followed by centrifugation at 12,000×g, 4℃ for 10 min, and the supernatant was collected. Same amount of protein sample was separated by SDS–polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk solution for 2 hours, then incubated with primary antibodies (listed in Supplementary Table 2) at 4℃ overnight. After thrice washing for 10 minutes with TBST, the membranes were incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:10000) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (= 1:20000) for 1.5 hours at room temperature. The target proteins were visualized using an enhanced chemiluminescence substrate and chemiluminescence imaging system. Band intensities were detected and analyzed using image J software, Version 1.8.0.112 (National Institutes of Health (NIH)). Quantitative PCR ( qPCR) The extraction of total RNA utilized the HiPure Total RNA Mini Kit, followed by reverse transcription using the Reveaid First Strand cDNA Synthesis Kit. For qPCR analysis, 2 µl of the resulting cDNA underwent amplification and measurement employing the Power Sybr Green PCR Master Mix. Reactions were executed on a Bio-Rad CFX Maestro 1.0 ABI PRISM 7300 real-time cycler (Applied Biosystems, Foster City, CA). The protocol included a melting step at 95°C for 10 minutes, followed by 49 amplification cycles (95°C, 10 s; 55°C, 30 s; 65°C, 5 s). mRNA levels for each gene were determined utilizing the ΔΔCT method with GAPDH serving as a reference. The primers utilized for RT-PCR are detailed in Table 4. Transmission electron microscope Liver tissue specimens were meticulously processed to ensure accurate characterization of microstructural alterations underlying metabolic associated fatty liver disease (MAFLD) pathogenesis. Initially, liver samples were meticulously fixed in 2.5% glutaraldehyde solution. Subsequently, a systematic dehydration process was employed utilizing a graded series of ethanol concentrations ranging from 30–100%, facilitating the removal of water from the tissue while maintaining structural integrity. Following dehydration, the specimens underwent critical point drying to further eliminate residual moisture. To enhance electron conductivity and imaging quality, the dehydrated liver samples were coated with a thin layer of platinum via sputtering. Ultrastructural observations of liver microarchitecture were performed utilizing a transmission electron microscope (JEM-1400, Japan). Statistical analysis Quantitative data were presented as mean values accompanied by standard error of mean (SEM). Statistical comparisons were conducted employing rigorous analytical methods, including one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-hoc test to determine intergroup differences. Significance levels were set at P < 0.05, ensuring robust statistical evaluation and interpretation of experimental findings. Results Influence of EA on glycolipid metabolism and insulin resistance in T2DM model rats Figure 1 A, B depicted significant weight and FBG level increases in T2DM rats before intervention, confirming successful T2DM model establishment. Throughout the study, FBG levels in the T2DM group continued to rise, significantly peaking post-intervention. Compared to pre-intervention levels, FBG concentrations in EA, Met, and EA + Met groups notably decreased after the fourth, fifth, and sixth weeks of treatment. However, the FBG levels in the EA + Met group, although the lowest, did not show statistical significance compared to the other groups. Conversely, FBG levels in the EA + Compd C group were markedly higher than those in the EA group post-intervention. FBG levels in the Con group remained within the normal range throughout the study. Following intervention, T2DM rats exhibited insulin resistance and lipid metabolism disturbances. Serum TC, TG, LDL, weight, and HOMA-IR levels significantly surpassed those of the Con group, while serum HDL levels and HOMA-ISI notably decreased in the T2DM group. Conversely, all treatment groups showed significant reductions in TC, TG, LDL, weight, and HOMA-IR levels, with notable increases in HDL levels and HOMA-ISI (Fig. 1 B-D) The phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT/PKB) signaling pathway / glucose transporter-4 (GLUT4) signaling pathway, crucial for insulin-stimulated glucose transport, showed suppression in the T2DM group compared to the Con group in the results of Western Blotting. However, EA, Met, and EA + Met treatments reversed the downregulation of hepatic PI3K, AKT, and GLUT4 protein expression. While the EA + Met group exhibited greater increases in protein expression, the differences were not statistically significant. (Fig. 2 B, D). AMPK, a key regulator of fatty acid, glucose homeostasis, and insulin sensitivity, was investigated along with its downstream signaling pathways PPARα/CPT1A, SREBP1c and PGC-1α/PCK2, which responsible for gluconeogenesis, fat generation, and fatty acids oxidative utilization. HFD + STZ treatment led to alterations in protein expression levels, which were reversed by EA, Met, and EA + Met treatments. qPCR results further confirmed these findings, showing significant changes in mRNA levels of related genes. The results showed that PPARα and CPT1A protein expression were distinctly suppressed, while PGC-1α and PCK2 protein expression were distinctly upgraded by HFD + STZ treatment as compared to the Con group. However, EA, Met and EA + Met treatment reversed the changes of PPARα, CPT1A, PGC-1α and PCK2 protein expression in T2DM rats (Fig. 5 A, C). The results of qPCR further confirmed that the mRNA levels of PGC-1α and SREBP-1c were significantly increased in T2DM group and decreased by EA, Met and EA + Met treatment, meanwhile the mRNA levels of PPARα were decreased in T2DM group and increased in EA, Met and EA + Met groups (Fig. 5 C). We further performed intraperitoneal injections of the AMPK inhibitor (Compound C) in T2DM rats treated with EA, to investigate the target of EA in the treatment of T2DM. The results revealed increased levels of FBG, TC, TG, LDL, HOMA-IR, and decreased HDL and HOMA-ISI compared with the EA group. Additionally, the expression of PI3K/AKT/GLUT4, PPARα/CPT1A signaling pathways was down-regulated, while PGC-1α/PCK2 signaling pathway and SREBP1c expression was upgraded in the EA + Compd C group compared to the EA group. EA-mediated autophagy ameliorated hepatic function and morphological change in T2DM rats. As shown in Fig. 3 A, histopathological analysis (H&E staining) revealed evident liver damage in T2DM rats, characterized by hepatocyte swelling, lipid vacuoles, ballooning, inflammation, and lobular structure irregularities. Notably, after 6 weeks of EA and Met treatment, these pathological features improved significantly, with EA + Met nearly restoring hepatocyte morphology to normal levels, indicating a potent synergistic effect of the two interventions. Furthermore, staining with Oil Red O (Fig. 3 B), a dye commonly used to visualize lipid droplets, demonstrated substantial lipid accumulation in T2DM rat livers compared to the Con group, which decreased significantly after EA and Met treatment, suggesting an attenuation of hepatic steatosis—a prevalent complication in T2DM-associated liver dysfunction. The combined group exhibited lower lipid accumulation than the EA or Met groups. Furthermore, serum AST, ALT, and ALB levels, the critical hepatic function indicators, were used to appraise the hepatoprotective effect of EA, Met and EA + Met in T2DM rats (Fig. 3 C). In rats with T2DM, serum AST and ALT levels exhibited a decline, concomitant with an elevation in serum ALB levels. Upon administering three distinct treatments (EA, Met, EA + Met), the trend in AST and ALT levels showed a obvious decrease, whereas ALB levels experienced a subsequent increase. Remarkably, the combined approach of EA alongside medication demonstrated superior efficacy in modulating ALT levels compared to both metformin monotherapy and EA interventions. This finding not only underscored the potential synergistic effects of combining therapeutic modalities but also highlighted EA's promising role in ameliorating hepatic function in the context of T2DM. The role of autophagy in T2DM-induced MAFLD emerges as pivotal, serving as a cellular mechanism that mitigates liver steatosis and maintains cellular homeostasis. Transmission electron microscopy revealed pronounced differences between the control and T2DM groups in liver cell composition. In the T2DM group, substantial lipid droplets were evident alongside blurred mitochondrial structure, expanded endoplasmic reticulum, and diminished autophagosomes, whereas EA, Met, and EA + Met groups showed restored cellular morphology with increased autophagosomes and reduced lipid droplets (Fig. 3 D). The autophagy activity in liver of T2DM rats was examined by western blotting, qPCR, immunohistochemistry and immunofluorescence. In Fig. 4 A-C and F-G, a notable downward trend in both protein and mRNA expression of LC3II was observed in the T2DM group, indicating suppressed autophagy activity. Conversely, the EA, Met, and EA + Met groups exhibited a significant upsurge in LC3II expression, suggesting that EA, Met, and EA + Met interventions partially restored autophagy activity impaired by T2DM. Additionally, to delve deeper into EA's mechanism of action in combating diabetes by ameliorating hepatic lipid accumulation through autophagy, we scrutinized the effects of the AMPK/mTOR pathway in the livers of T2DM rats. The results from Fig. 5 A-E and G indicated that HFD + STZ treatment significantly increased p-mTOR/mTOR and mTOR mRNA levels, while decreasing p-AMPK/AMPK and AMPK mRNA levels. Conversely, EA, Met, and EA + Met treatment restored these levels. This suggests that EA may regulate liver autophagy activity through the AMPK/mTOR pathway. Immunohistochemical analysis mirrored the western blotting and qPCR findings for AMPK and mTOR expression in the liver. Notably, the EA + Met group exhibited notably superior hepatic AMPK expression compared to the EA and Met groups (Fig. 5 G). The design of the combination of EA and AMPK inhibitor group further validated the above conclusion. This study also showed that Compound C effectively inhibited the promotion of autophagy by EA in liver, including a marked augmented level of mTOR and low expression of AMPK and LC3II, which could explain the increase of lipid droplet, the decrease of autophagosomes, hepatic morphological damage, and reduced liver fuction. EA reduces liver fibrosis induced by TGF β1/smad3 pathway Specific stains, like Masson staining, were used to detect collagenous fibers and evaluate hepatic fibrosis. In Fig. 5 D, the Masson staining revealed a stark contrast between the normal and model groups in terms of hepatic fibrosis, with the model group displaying a significant increase in collagen fibers. Conversely, treatment with EA, Met, and EA + Met notably reversed the presence of blue collagen fibers, particularly notable in the EA + Met group. These findings indicated that EA effectively mitigated T2DM-induced hepatic fibrosis. Transmission electron microscopy revealed significant collagen fibers in the perisinusoidal space of the liver in the T2DM group, a feature notably reduced in the EA, Met, and EA + Met groups (Fig. 5 H). The immunofluorescence results had showed that the model group exhibited increased α-SMA expression compared to the Con group. However, in the EA, Met, and EA + Met groups, α-SMA expression decreased significantly compared to the model group, indicating three treatment’s potential to inhibit hepatic fibrosis. This was further supported by Western blot analysis (Fig. 5 A-C, F-G). The association of the TGFβ1/Smad3 pathway with liver fibrosis has been extensively demonstrated. To investigate whether the protective effect of EA involves inhibition of the TGFβ1/Smad3 pathway, we assessed changes in this pathway using Western blotting, immunohistochemistry, and qPCR methods. Primarily, Western blot and immunohistochemistry analyses revealed significantly elevated expressions of TGFβ1 and Smad3 in the T2DM group compared to the control group (Fig. 5 A, B, and C). However, after 6 weeks of EA, Met, and EA + Met treatment, these expressions were notably suppressed. Furthermore, qPCR results mirrored these findings: liver tissue from T2DM rats exhibited increased mRNA expression of TGFβ1, Smad2, and Smad3 (Fig. 5 C). Yet, treatment with EA, Met, and EA + Met effectively inhibited the mRNA expression of TGFβ1, Smad2, and Smad3. To further identify the involvement of glycolipid metabolism and autophagy mediated by AMPK in the formation of liver fibrosis, the therapeutic action of EA was further evaluated using intraperitoneal injection of compound C. The results indicated that the expression of TGFβ1, smad2/3, and α-SMA were significantly increased after EA + Compd C treatment compared with the EA group. Masson staining and transmission electron microscopy showed that the EA + Compd C group exhibited greater degree of collagenous fiber compared with the EA group (Fig. 5 A-C, E, G). Discussion Individuals diagnosed with T2DM are susceptible to hepatocyte injury and fibrosis, even when presenting with isolated steatosis in the initial stages. This progression underscores the significance of managing T2DM-induced hepatic steatosis to forestall the development of fibrosis and the onset of MAFLD. The presence of liver fibrosis, often a precursor to cirrhosis, is notably more prevalent among individuals with diabetes, highlighting the urgent need for effective therapeutic interventions ( 48 , 49 ). However, the current clinical landscape is marked by a dearth of FDA-approved drugs specifically tailored for treating MAFLD ( 32 ). Addressing this gap, the present study delves into the protective potential of EA against T2DM-induced hepatic steatosis and ensuing fibrosis. Utilizing a T2DM rat model, this investigation scrutinizes the comparative efficacy of EA, metformin, and a combination thereof in mitigating the progression of hepatic complications. Metformin, a cornerstone medication in T2DM management, operates through the activation of the AMPK pathway, thereby enhancing liver function and insulin sensitivity ( 50 , 51 ). The study endeavored to elucidate whether the combined administration of EA and metformin yields synergistic effects, potentially offering a novel therapeutic approach for ameliorating hepatic dysfunction in T2DM. Our studies indicated that hyperglycemia, hyperlipidemia and IR induced by high-fat diet (HFD) and injection of STZ promoted lipid accumulation, pathological and functional damage, and resulting in hepatic steatosis and fibrosis. Meanwhile, we found that EA, Met, and EA + Met enhanced the activation of AMPK, which in turn promoted the expression of PPARα/CPT1A pathway, inhibited the expression of SREBP1c and PGC-1α/PCK2 pathway, futher improved levels of autophagy mediated by AMPK/mTOR pathway and thus suppressed TGFβ1/Smad2/3 signaling pathway, ultimately exerting its effect on ameliorating hepatic steatosis and fibrosis in T2DM rats. In a nutshell, our study revealed that the three treatment prominently alleviated T2DM-induced MAFLD, the mechanism was likely associated with the regulation of glycolipid metabolism and autophagy mediated by AMPK signaling pathway in liver. The interplay between T2DM and MAFLD entails intricate bidirectional regulation mechanisms, influencing each other's progression and severity ( 52 ). T2DM and IR instigate hepatic triglyceride accumulation, exacerbating MAFLD, while lipid toxicity and hyperglycemia further fuel the development of MAFLD ( 52 ). Conversely, the presence of MAFLD aggravates insulin resistance, establishing a detrimental cycle of mutual reinforcement. The AMPK pathway emerges as a pivotal player in bridging the pathophysiological connection between T2DM and MAFLD, orchestrating glycolipid metabolism and insulin resistance ( 33 , 36 ). In our study using T2DM rats induced by HFD + STZ treatment, we observed that intervention with EA treatment in T2DM rodent models exhibited hepatoprotective effects by bolstering AMPK activity. Numerous studies emphasized AMPK's pivotal role in T2DM and MAFLD development and progression ( 53 ). AMPK, comprising one catalytic subunit (α1 or α2) and two regulatory subunits, serves as a vital regulator of energy balance in liver, muscle, and adipose tissues ( 54 – 56 ). AMPK conserves ATP and modulates energy metabolism by activating catabolic pathways and inhibiting anabolic pathways, including fatty acid oxidation, hepatic lipogenesis, glucose uptake and output, and insulin sensitivity regulation ( 36 ). AMPK phosphorylation inhibits transcription factors that induce gluconeogenesis and lipogenic programs, notably PCK2 and SREBP1, reducing liver glucose and fat accumulation ( 33 , 57 ). Additionally, AMPK promotes fatty acid entry into mitochondria and oxidation through the CPT1 system via PPARα ( 37 ), as shown in Fig. 6 . Regular exercise enhanced whole-body insulin sensitivity by activating AMPK, as evidenced by epidemiological data indicating lower prevalence of metabolic syndrome diseases among physically active individuals ( 58 , 59 ). Strategies aimed at activating AMPK hold significant promise for both the prevention and treatment of T2DM and MAFLD. To delve deeper into the mechanism underlying EA, we conducted experiments utilizing T2DM rat models subjected to EA treatment, assessing the expression of molecular markers associated with AMPK. Throughout our rigorous investigation, stark disparities were observed between the control group and the T2DM rats. Specifically, levels of FBG, serum lipid components (including TG, TC, LDL), and HOMA-IR significantly escalated in the T2DM group. Conversely, serum HDL and HOMA-ISI levels notably declined. These findings underscored the manifestation of evident IR and dysregulation in glycolipid metabolism within the T2DM rat models. Following a 6-week intervention period, all three treatment modalities not only induced a substantial decrease in FBG levels from the fourth to sixth week but also precipitated a remarkable reduction in TC, TG, LDL, and HOMA-IR levels, coupled with a significant elevation in HDL and HOMA-ISI levels. These salutary effects were consonant with the activation of AMPK, culminating in the upregulation of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), and glucose transporter type 4 (GLUT4), alongside the inhibition of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), phosphoenolpyruvate carboxykinase 2 (PCK2), and sterol regulatory element-binding protein 1c (SREBP1c). Consequently, these cascades led to diminished adipogenesis and gluconeogenesis, augmented fatty acid oxidation, and heightened insulin sensitivity, thereby substantiating the therapeutic potential of AMPK activation in ameliorating the metabolic derangements associated with T2DM and MAFLD. The regulatory impact of EA, when combined with Met intervention, on the expression of hepatic AMPK surpassed that of both EA and Met administration. This superiority underscored the potential advantage of integrating EA with Met therapy in clinical practice. To assess the impact of enhanced glycolipid metabolism on the liver, we examined liver function and pathological changes. Our observations revealed elevated levels of ALT and AST, alongside reduced ALB levels in T2DM rats compared to the control group. Notably, the livers of T2DM rats exhibited inflammation, lipid vacuoles, ballooning, and significant fat accumulation, indicating liver metabolic dysfunction and hepatocyte steatosis. Furthermore, our results indicated that the three treatments effectively alleviated liver injury induced by IR. This was evidenced by decreased AST and ALT levels, increased ALB levels in serum, and improvements in hepatic morphology, along with reduced areas of positive Oil Red O staining. Moreover, electron microscope analysis revealed a notable presence of autophagosomes in liver tissue across the three treatment groups. Autophagy, an essential system for maintaining cellular homeostasis, mitigates liver injury, oxidative stress, and inflammation by breaking down and recycling misfolded proteins and damaged organelles resulting from lipid peroxidation. This process thereby prevents the onset of hepatic steatosis ( 24 ) ( 28 ). Lipophagy, a subtype of autophagy, targets intracellular lipid droplets (LDs) for degradation, aiming to regulate liver fat stores ( 25 , 26 , 60 ). Research indicated that compromised autophagic flux contributed to lipid droplet accumulation in hepatocytes, exacerbating hepatic steatosis and damage in T2DM rats. LC3-II, a protein crucial for autophagosome formation and maturation, serves as a reliable marker for assessing autophagic activity ( 61 , 62 ). In the present study, both the protein and mRNA expression levels of LC3-II were significantly reduced in the T2DM group, suggesting suppressed autophagic activation. This finding correlated with the observed increase in hepatic lipid deposition and morphological alterations. Conversely, LC3B-II levels were markedly elevated, accompanied by a reduction in LDs following the three treatments compared to the T2DM group. This suggested that enhanced autophagy facilitated lipid clearance in the liver. Consequently, EA, Met, and EA + Met treatments may alleviate hepatic lipid accumulation by inducing autophagy in T2DM. Central to the regulation of autophagy in MAFLD is the AMPK /mTOR pathway, which has been extensively documented ( 63 ). AMPK, a highly conserved serine/threonine-protein kinase across evolutionary scales, serves as a pivotal energy sensor, thereby regulating metabolic homeostasis and amplifying autophagy ( 38 , 39 ). Conversely, mammalian target of rapamycin (mTOR), a central regulator of cell growth, inhibits autophagy by integrating nutrient signals and growth factors. mTORC1, a complex of mTOR ( 64 ), phosphorylates ULK1 at Ser757, impeding its interaction with AMPK, thus halting autophagy. AMPK, as the upstream regulator of mTOR, negatively modulates mTOR activation either directly by phosphorylating mTORC1 or by phosphorylating various components of mTORC1. Moreover, AMPK fosters autophagy by directly activating ULK1 through phosphorylation at Ser555 and Ser777, facilitating the release of ULK1 from mTORC1. Consequently, the activated ULK1 kinase complex recruits other autophagy proteins, instigating autophagy induction ( 63 , 65 – 68 ). However, in conditions such as steatosis and hypernutrition, autophagy initiation is hindered due to alterations in AMPK and mTOR signaling. The current study revealed a significant decrease in the p-AMPK/AMPK ratio and AMPK mRNA alongside a remarkable increase in the p-mTOR/mTOR ratio and mTOR mRNA. This was coupled with the inhibition of autophagy, as evidenced by the notable decrease in LC3-II levels in the T2DM group. However, autophagy was restored in the groups treated with EA, Met, and EA + Met, achieved through elevated AMPK expression and reduced mTOR expression levels, consequently leading to increased LC3-II levels. Moreover, our results showed that EA-induced autophagy and improvement of hepatic lipid droplet accumulation and glycolipid metabolism was partly disrupted by Compound C, an inhibitor of AMPK, in the EA + Compd C group. Taken together, these results suggest that the beneficial effects of EA may stem from enhanced autophagy via the AMPK/mTOR pathway. Chronic liver injury caused by T2DM and metabolic disorders gradually progresses to fibrosis, which is characterized by excess deposition of collagen between hepatocytes and hepatic sinusoids to repair the liver damage ( 69 ). Collagen, a pivotal structural protein within the extracellular matrix (ECM), serves as a fundamental component in maintaining tissue integrity and function. In the liver, hepatic stellate cells (HSCs) emerge as the primary source of collagen synthesis, playing a critical role in the dynamic equilibrium of ECM turnover and regulation. In response to liver injury, HSCs undergo a phenotypic transformation into activated myofibroblast-like cells (MFCs), which are proficient in ECM production, notably collagen types I and III, and formate α-smooth muscle actin (α-SMA) stress fibers ( 70 , 71 ). This transition marks a pivotal event in the pathogenesis of liver fibrosis, characterized by excessive ECM deposition and aberrant tissue remodeling. Central to the fibrotic cascade is the multifaceted cytokine transforming growth factor-beta (TGF-β), which orchestrates various cellular processes, including HSCs activation and proliferation. Within the liver microenvironment, TGF-β1 emerges as a key isoform, synthesized by both liver parenchymal cells and activated HSCs, exerting its effects through a Smad3-dependent signaling pathway ( 72 , 73 ). Upon binding to the TGF-β II receptor on the cell membrane of HSCs, TGF-β1 initiates a signaling cascade culminating in the activation of intracellular mediators, notably Smad proteins ( 74 ). Subsequently, the Smad complex, comprising Smad2, Smad3, and Smad4, translocates into the nucleus, where it modulates gene transcription, including the upregulation of collagen expression. By directly binding to the collagen promoter region, the Smad complex exerts transcriptional control, thereby fostering ECM production and deposition, thereby perpetuating the fibrotic response in the liver ( 75 , 76 ). Understanding the intricate interplay between HSCs, TGF-β signaling, and collagen synthesis offers crucial insights into the molecular mechanisms underpinning liver fibrosis. Targeting these pathways holds promise for the development of novel therapeutic strategies aimed at mitigating fibrotic progression and restoring hepatic homeostasis. Our results showed that the expressions of α-SMA, a fibroblast marker, were abundantly increased in the T2DM group. The pathological characteristics of T2DM in massson staining and electron-microscope exhibited an increase in hepatic fibrosis, indicating activation of HSCs. We also found that HFD + STZ treatment distinctly increased the expressions of TGFβ1, smad2, and smad3. These data prompted that T2DM-induced aberrant glycolipid metabolism possess a critical role in liver injury and fibrosis in T2DM rats. It is worth mentioning that the three treatments could attenuated this effect and significantly ameliorate hepatic fibrosis and decrease the expression of TGFβ1, smad2, and smad3 in T2DM rats, suggesting that the antifibrotic activity of three treatments may be associated, at least partially, with TGFβ1/smad2/3 signaling pathway activity attenuation. However, there arised a question: was the upregulation of autophagy and glycolipid metabolism by EA linked to the downregulation of TGFβ1/SMAD3 signaling, or was it an independent molecular event requiring further investigation? As previously mentioned, AMPK-mediated glycolipid metabolism and autophagy contributed to restoring liver structure and function, thereby preventing liver fibrosis by mitigating factors causing liver damage. Rangnath Mishra investigated the interplay between AMPK and the TGFβ1/SMAD3 pathway. Their study revealed that pharmacologically activating AMPK inhibited TGFβ-induced secretion of collagen types I and IV and fibronectin, driven by Smad3-binding cis-elements. In our present study, as anticipated, the introduction of Compound C, an AMPK inhibitor, suppressed AMPK activation and partially reversed the beneficial effects of EA on the TGFβ1/SMAD3 signaling pathway and liver fibrosis. This finding confirmed a direct relationship between AMPK activation and the anti-fibrotic effects of EA treatment. Considering the aforementioned studies, the AMPK signaling pathway mediatedEA's beneficial effects in delaying the progression of MAFLD in T2DM rats. In conclusion, to the best of our knowledge, the results of the present study showed that EA played a critical role in ameliorating in MAFLD in T2DM rats. Specifically, EA intervention improved FBG, serum lipids, insulin sensitivity and hepatic function, and inhabited liver steatosis and fibrosis of T2DM rats. Furthermore, we demonstrated insights that EA treatment was likely through the mechanisms of the up-regulating of hepatic AMPK signaling pathway, which mediated glycolipid metabolism and autophagy, inhibited TGFβ1/SMAD3 pathway-induced fibrosis. Moreover, the above effects of EA were consistent with metformin. The combination of EA and metformin had significant advantages in increasing hepatic AMPK expression, improving liver morphology, lipid droplet infiltration, fibrosis, and reducing serum ALT levels. Taken together, our findings indicated that EA might be taken as an effective therapeutic strategy and improving the efficacy of metformin in MAFLD. Therefore, the combined approach of EA and Met in clinical practice offers a personalized and holistic treatment strategy that considers the individual's constitutional differences and the dynamic nature of metabolic disorders. Through synergistic interactions, acupuncture may enhance the efficacy of pharmacotherapy while minimizing adverse effects and promoting overall well-being. Future research endeavors should focus on elucidating the underlying mechanisms responsible for the superior regulatory effect of EA combined with Met on liver AMPK expression. Additionally, multiple animal studies and larger clinical trials are warranted to validate the therapeutic efficacy and safety of this integrated approach in diverse patient populations with metabolic disorders. Ultimately, integrating EA with Met holds promise for advancing precision medicine and improving patient outcomes in the management of metabolic diseases. Declarations Author Contributions HD: Conceptualization, Writing—original draft, Project administration. SS: Data curation, Methodology. RL: Supervision, Funding acquistion. SH: Writing – review & editing. SZ: Visualization. SL: Software. XL: Formal analysis. WG: Validation. Funding This study was supported by the following grants: 1. National Natural Science Foundation of China (Grant Nos. 81973935); 2. Beijing Municipal Natural Science Foundation (Grant Nos. 7232276). Acknowledgments The authors thank the School of Acupuncture - Moxibustion, and Tuina, Beijing University of Chinese Medicine for supplied experimental equipment and research environment. Ethical Approval The protocol received approval from the Laboratory Animal Welfare and Ethics Committee of Beijing University of Chinese Medicine (BUCM-4-2021051301-2026). 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Supplementary Files SupplementaryMaterial.docx Table1.xls Table2.xls Table3.xls Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Diabetology & Metabolic Syndrome → Version 1 posted Editorial decision: Revision requested 15 Jun, 2024 Reviews received at journal 15 Jun, 2024 Reviews received at journal 15 Jun, 2024 Reviewers agreed at journal 08 Jun, 2024 Reviewers agreed at journal 05 Jun, 2024 Reviewers invited by journal 29 May, 2024 Editor assigned by journal 29 May, 2024 Submission checks completed at journal 28 May, 2024 First submitted to journal 25 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4475748","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312074539,"identity":"cf9bdc3a-7e83-4ba9-b179-26dc73e68297","order_by":0,"name":"Haoru DUAN","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haoru","middleName":"","lastName":"DUAN","suffix":""},{"id":312074540,"identity":"3e0289dd-e41f-4933-a61a-301d84b25557","order_by":1,"name":"Shanshan Song","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Song","suffix":""},{"id":312074541,"identity":"57ead8ab-4a8a-4471-9483-1918e28f16d7","order_by":2,"name":"Rui Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACZihtwMDY+CChwoawDh4kLc0GD86kEaEFxjBgYGCTfNh2iLAWe3bew69522zszdkPt1UksB1g4G/vTiDgML40y5ltacyWPYltNxJ47jBInDm7gYAWHjODj22H2QwOgLRIPGMwkMglQkti238eg/MP2woSDA4TpcX4wce2AxIGNxLbGBISiNFymMeMcca5ZAODGw+bJRIOpPEQ9At7/xnjzzxldvYG59Mffvz5z0aOv70XvxYgYJNAsZaQchBg/kCMqlEwCkbBKBjBAADT6kZjXPjTygAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Rui","middleName":"","lastName":"Li","suffix":""},{"id":312074542,"identity":"bce9a945-5596-40c8-b131-42131bb71836","order_by":3,"name":"Suqin Hu","email":"","orcid":"","institution":"Henan Province Hospital of Traditional Chinese Medicine, Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Suqin","middleName":"","lastName":"Hu","suffix":""},{"id":312074543,"identity":"ed460dc5-ec21-45b9-b6fa-41935b37865c","order_by":4,"name":"Shuting Zhuang","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Zhuang","suffix":""},{"id":312074544,"identity":"8c0352a3-2425-4552-befb-639e8caddb74","order_by":5,"name":"Shaoyang liu","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shaoyang","middleName":"","lastName":"liu","suffix":""},{"id":312074545,"identity":"0048389b-04c5-4713-9d24-2a62e4eb932b","order_by":6,"name":"Xiaolu Li","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaolu","middleName":"","lastName":"Li","suffix":""},{"id":312074546,"identity":"99693f03-ce9c-4be9-a5b0-8774525f19f0","order_by":7,"name":"Wei Gao","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-05-25 07:55:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4475748/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4475748/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13098-024-01432-7","type":"published","date":"2024-09-11T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58055791,"identity":"9a6a5532-a8c3-49af-b43b-5c41f0947e2b","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":121022,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of EA on the levels of FBG, serum lipid, and insulin in T2DM model rats. A: Fasting blood glucose, B:Weight, C: TG, TC, LDL, HDL, D: insulin (INS), insulin resistance index (HOMA-IR) and insulin sensitivity index (HOMA-ISI). Con: the control group, T2DM: the T2DM group, EA: the electroacupuncture group, Met: the metformin group, EA+Met: combination group of electroacupuncture and metformin, EA+Compd C: combination group of electroacupuncture and compound C. Data are expressed as mean ± standard error of mean (n = 10 rat per group). Compared with the Con group, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the T2DM group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA group, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the same group before intervention, \u003csup\u003e●\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e●●\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/7e65b3f29828cc699095fd98.png"},{"id":58056697,"identity":"74f48385-b7cf-409c-84ee-93b8e4bad026","added_by":"auto","created_at":"2024-06-10 14:23:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":290252,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of EA on hepatic glycolipid metabolism and insulin resistance in T2DM model rats. A: Protein expression of hepatic glycolipid metabolism signaling pathways. B: Protein expression of hepatic insulin signaling transduction pathway. C: (1)-(4) Quantification of protein levels of PPARα, CPT1A, PGC1-α, PCK2 (n = 3 per group), (5)-(7) mRNA levels of PPARα, SREBP1c, PGC1-α (n = 6 per group). D: Quantification of protein levels of PI3K, AKT, GLUT4 (n = 3 per group). Con: the control group, T2DM: the T2DM group, EA: the electroacupuncture group, Met: the metformin group, EA+Met: combination group of electroacupuncture and metformin, EA+Compd C: combination group of electroacupuncture and compound C. Data are expressed as mean ± standard error of mean. Compared with the Con group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the T2DM group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA group, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/ae1edf5758e40b69549e2e87.png"},{"id":58055796,"identity":"3f29809c-a2ac-447e-ac02-fe8703758147","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":597882,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of EA on the morphology and function of the liver in T2DM model rats. A: HE staining showed hepatic histological changes after intervention (red arrow: hepatocytic lipid vacuoles, black arrow: hepatocyte ballooning). Images are shown at the original magnification of 400×. B: Oil Red O staining evaluated liver steatosis (red arrow: lipid droplets). Images are shown at the original magnification of 400×. C: levels of serum AST, ALT, ALB (n = 10 per group). D: Electron microscopy observed autophagy and lipid droplets in liver cells (red arrow: autophagosomes, yellow arrow: lipid droplets, blue arrow: mitochondria, purple arrow: endoplasmic reticulum, green arrow: lipid vacuoles). Images are shown at the original magnification of 15000×. Con: the control group, T2DM: the T2DM group, EA: the electroacupuncture group, Met: the metformin group, EA+Met: combination group of electroacupuncture and metformin, EA+Compd C: combination group of electroacupuncture and compound C. Data are expressed as mean ± standard error of mean. Compared with the Con group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the T2DM group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA group, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA+Met group, \u003csup\u003e●\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/7922dcb09af323061bc5f666.png"},{"id":58055797,"identity":"93ae76fc-dc5d-43a9-a0da-3ba14537a82f","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2249914,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of EA on hepatic autophagy in T2DM model rats. A: Protein expression of hepatic autophagy signaling pathway. B: Quantification of protein levels of p-AMPK/AMPK, p-mTOR/mTOR, LC3Ⅱ (n = 3 per group). C: mRNA levels of AMPK, mTOR, LC3Ⅱ (n = 6 per group). D, E: Representative images of immunohistochemical staining of AMPK, mTOR expressions in the liver. Images are shown at the original magnification of 400×. F: Representative images of immunofluorescence staining of LC3Ⅱ expressions in the liver. Images are shown at the original magnification of 400×. G: Quantitative analyses according to groups (n = 6 per group). Con: the control group, T2DM: the T2DM group, EA: the electroacupuncture group, Met: the metformin group, EA+Met: combination group of electroacupuncture and metformin, EA+Compd C: combination group of electroacupuncture and compound C. Data are expressed as mean ± standard error of mean. Compared with the Con group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the T2DM group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA group, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA+Met group, \u003csup\u003e●\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e●●\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/8b5f7b6af216205388fee34f.png"},{"id":58055800,"identity":"f9e13d97-14bd-424f-a13e-df3dffdb8eda","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2589666,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of EA on liver fibrosis in T2DM model rats. A: Protein expression of hepatic fibrosis signaling pathway. B: Quantification of protein levels of TGFβ1, SMAD3, α-SMA (n = 3 per group). C: mRNA levels of TGFβ1, SMAD2, SMAD3 (n = 6 per group). D: Masson staining evaluated liver fibrosis (black arrow: collagen fibers). Images are shown at the original magnification of 400×. E: Representative images of immunohistochemical staining of TGFβ1 expressions in the liver. Images are shown at the original magnification of 400×. F: Representative images of immunofluorescence staining of α-SMA expressions in the liver. Images are shown at the original magnification of 400×. G: Quantitative analyses according to groups (n = 6 per group). H: Electron microscopy observed collagen fibers in liver cells (white arrow: collagen fibers). Images are shown at the original magnification of 15000×. Con: the control group, T2DM: the T2DM group, EA: the electroacupuncture group, Met: the metformin group, EA+Met: combination group of electroacupuncture and metformin, EA+Compd C: combination group of electroacupuncture and compound C. Data are expressed as mean ± standard error of mean. Compared with the Con group, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the T2DM group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the EA group, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/dbd93237423bc06006fc09c8.png"},{"id":58055799,"identity":"e854b149-7537-4a18-a945-c453ed3f48ab","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":757824,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between AMPK and metabolic signaling pathways.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/a951c12347c944e89c9dcae8.png"},{"id":64619138,"identity":"791ed989-7881-4b5f-b0f2-fed3e26301ea","added_by":"auto","created_at":"2024-09-16 16:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8054685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/43f0a7b2-de00-4c55-9221-9bf95ec1457e.pdf"},{"id":58055801,"identity":"1a1cb256-dfeb-44ec-bfdd-3861ff3c1883","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29760762,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/aa670674ee2cf6714350d26e.docx"},{"id":58055798,"identity":"b24517fb-75c0-4fa6-b104-1021e93d7777","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20480,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xls","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/4dfa3859e65a4d43591f06a2.xls"},{"id":58056168,"identity":"2ed87c46-5e09-448c-b575-40f107770527","added_by":"auto","created_at":"2024-06-10 14:15:00","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21504,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xls","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/6113390aaf68c9518985f520.xls"},{"id":58055793,"identity":"5e8f459f-8c85-4faf-9dab-f1467f15e692","added_by":"auto","created_at":"2024-06-10 14:07:00","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20480,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.xls","url":"https://assets-eu.researchsquare.com/files/rs-4475748/v1/68c5537e0e7211e12db7bb21.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Strategy for treating MAFLD: Electroacupuncture alleviates hepatic steatosis and fibrosis by enhancing AMPK mediated glycolipid metabolism and autophagy in T2DM rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNon-Alcoholic Fatty Liver Disease (NAFLD), recently redefined as Metabolic Associated Fatty Liver Disease (MAFLD), presents a significant health burden worldwide, particularly among individuals with Type 2 Diabetes Mellitus (T2DM). The progression of MAFLD from simple hepatic steatosis to steatohepatitis and fibrosis poses substantial risks to affected individuals, particularly those with comorbid T2DM (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Studies had consistently shown that T2DM patients faced a substantially elevated risk up to 70% \u0026minus;\u0026thinsp;80% of developing MAFLD compared to non-diabetic counterparts (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Importantly, the convergence of T2DM and MAFLD escalated the risk of cardiovascular complications due to compromised metabolic control (\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). A recent prospective case-control study involving 2103 T2DM patients underscored this risk, revealing an association between MAFLD and increased cardiovascular disease risk over a five-year follow-up period, even after adjustments for other pertinent risk factors were made (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Hence, the evaluation and management of MAFLD, even in its milder forms, are imperative for mitigating cardiovascular risk and improving overall mortality rates among individuals with T2DM.\u003c/p\u003e \u003cp\u003eThe pathological process of MAFLD is intricately intertwined with insulin resistance (IR), a condition characterized by decreased sensitivity of cells to the effects of insulin hormone. In MAFLD initiation, the primary manifestation is simple steatosis, characterized by the accumulation of triglycerides (TG) in over 5% of hepatocytes (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This accumulation is significantly influenced by IR, which serves as a driving force in the development and progression of MAFLD. One of the key consequences of IR is the increased influx of fatty acids into the liver, which, coupled with elevated hepatic de novo lipogenesis (DNL), results in heightened TG levels within hepatocytes (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Furthermore, inhibition of peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) reduces fatty acid oxidation (FAO), further contributing to TG accumulation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The failure of insulin to effectively regulate liver glucose uptake and production exacerbates hyperglycemia and IR, thereby exacerbating the pathogenic processes underlying MAFLD (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Persistent IR, coupled with increased free fatty acid (FFA) influx, induces mitochondrial dysfunction, reactive oxygen species (ROS) production, and toxic lipid accumulation, which in turn exacerbates hepatic damage (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). As MAFLD progresses, it encompasses fatty infiltration, lobular inflammation, and can manifest into severe forms such as non-alcoholic steatohepatitis (NASH) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). NASH, if left unchecked, may advance to perisinusoidal fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Given the reversible nature of MAFLD in its early stages, timely intervention becomes paramount in altering its trajectory and curbing its escalating prevalence (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Thus, there is an imperative need to address the underlying pathological factors, particularly focusing on interventions aimed at mitigating diabetes-induced liver injury.\u003c/p\u003e \u003cp\u003eAutophagy, a cellular process essential for maintaining homeostasis, serves as a protective mechanism against liver injury induced by various metabolic insults (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Specifically, in the context of MAFLD, dysregulated lipid metabolism and oxidative stress overwhelms cellular defenses, leading to hepatocyte dysfunction and the progression of liver pathology. Autophagy in hepatocytes plays a pivotal role in mitigating these effects by removing damaged organelles and lipid droplets, thus preventing excessive cell death and fibrosis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, when autophagy is impaired, as seen in MAFLD, the liver becomes more susceptible to injury and fibrotic progression (\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Studies have demonstrated that knocking out autophagy-related genes exacerbates hepatic fibrosis, underscoring the protective role of autophagy in liver pathology (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo optimize therapeutic outcomes in MAFLD, addressing metabolic disturbance, steatosis, and liver damage is crucial (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). One promising strategy for the treatment of T2DM-induced MAFLD involves targeting the AMP-activated protein kinase (AMPK) pathway, which plays a crucial role in cellular energy homeostasis and metabolism regulation (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Activation of AMPK has been shown to enhance glycolipid metabolism and autophagy, both of which are essential processes for mitigating the adverse effects of insulin resistance in MAFLD. By enhancing glycolipid metabolism, AMPK activation alleviates the excess accumulation of triglycerides in hepatocytes by promoting the conversion of glucose into energy rather than storing it as lipids (\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Additionally, autophagy induction facilitated by AMPK activation can aid in the removal of damaged mitochondria and toxic lipid species, thus mitigating the progression of hepatic damage in MAFLD (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Moreover, targeting AMPK signaling also improves insulin sensitivity, thereby addressing one of the central pathological features of MAFLD(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Therefore, interventions aimed at enhancing AMPK-mediated glycolipid metabolism and autophagy hold promise as potential therapeutic avenues for addressing insulin resistance in the pathological process of MAFLD. While several compounds are undergoing clinical investigation for MAFLD management, none have received specific approval yet (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Acupuncture, a unique external therapy in traditional Chinese medicine with a lengthy history of treating T2DM in China, has been reported to be effective against T2DM, IR, and diabetic liver injury (\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Recent studies, including our unpublished research, had indicated that EA's hypoglycemic and hypolipidemic effects protected the liver's morphology and function in T2DM rats by reducing inflammation and lipid droplet infiltration in liver tissue (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). These findings underscored EA's therapeutic potential in ameliorating T2DM with MAFLD, with the underlying molecular mechanism associated with AMPK activation. Building upon these promising results and \u0026ldquo;prevent before the disease exacerbats\u0026rdquo; thought in traditional chinese medicine (TCM), we hypothesized that EA might prevent hepatic steatosis and fibrosis in diabetes-induced hepatic dysfunction by enhancing the AMPK signaling pathway, resulting in the upregulation of glycolipid metabolism and autophagy and inhibiton of TGFβ/SMAD3 signaling pathways. Therefore, our present study primarily investigated EA's potential effect and mechanism of treatment and its specific relationship with hepatoprotection and anti-fibrosis in T2DM rats induced by a high-fat diet (HFD) and injection of STZ, aiming to provide therapeutic strategies for preventing or delaying the progression of MAFLD.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of T2DM animal model\u003c/h2\u003e \u003cp\u003eMale Wistar rats of Specific Pathogen Free (SPF) grade, weighing 150\u0026ndash;180 g, were procured from Sibeifu (Beijing) Biotechnology Co., Ltd. These rats were maintained at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, with a humidity of 40% \u0026plusmn; 5%, under a 12-hour light/dark cycle. After acclimatization for 7 days, 10 rats were randomly assigned to the Control group (Con), provided with ad libitum access to water and standard rat chow. The remaining rats were induced to develop a T2DM model through a high-fat diet combined with STZ injection. The induction protocol involved feeding the rats a high-fat diet (comprising 67% regular rodent chow, 20% sucrose, 10% lard, 2.5% cholesterol, and 0.5% sodium cholate) for 4 weeks, followed by an overnight fast of 20 hours, and subsequent intraperitoneal injection with a 2% solution of streptozocin (STZ) (dissolved in 0.1 mmol/L citrate buffer solution with a pH of 4.2\u0026ndash;4.5) at a dose of 35 mg/kg. The Con group rats were intraperitoneally injected with 0.1 mol/L pH\u0026thinsp;=\u0026thinsp;4.2 citric acid - sodium citrate buffer. The criterion for successful T2DM model preparation was set as Random Blood Glucose (RBG)\u0026thinsp;\u0026ge;\u0026thinsp;16.7mmol/L and/or Fasting Blood Glucose (FBG)\u0026thinsp;\u0026ge;\u0026thinsp;11.1 mmol/L. Throughout the 14-day model evaluation and 6-week interventions, rats, excluding those in the Con group, were fed a high-fat diet. A total of 60 rats were included as model animals, and they were randomly allocated into the following groups based on FBG levels: Control (Con), T2DM, Electroacupuncture (EA), Metformin (Met), Electroacupuncture combined with Metformin (EA\u0026thinsp;+\u0026thinsp;Met), and Electroacupuncture combined with Compound C (EA\u0026thinsp;+\u0026thinsp;compd C), each comprising 10 rats.\u003c/p\u003e \u003cp\u003eRats with EA treatment involved the use of disposable sterile acupuncture needles (0.13*7 mm) at bilateral acupoints, including Pishu (BL 19), Weiwanxiashu (EX B3), Zusanli (ST 36), and Sanyinjiao (SP 6). The needles were inserted to a depth of 4\u0026ndash;6 mm. Additionally, Weiwanxiashu (EX B3) and Zusanli (ST 36) were connected to an electroacupuncture instrument set to a continuous wave, with a frequency of 15 Hz and an amplitude of 2\u0026ndash;4 mA, for 20 minutes per session, Once a day. In contrast, rats in the Met group received a daily gavage of 60 mg/mL metformin solution, dosed at 5 mL/kg body weight. Meanwhile, rats in the EA\u0026thinsp;+\u0026thinsp;Met group underwent both EA and Met treatment once a day. The EA session lasted for 20 minutes, while Met was administered at a dosage of 300 mg/kg. Another group, the EA\u0026thinsp;+\u0026thinsp;compd C group, was subjected to intraperitoneal injection of Compound C at a dosage of 10 mg/kg, in addition to the daily 20-minute acupuncture session. Rats in the control and T2DM groups underwent immobilization similar to that of the EA group but without any other interventions. All interventions were carried out six times a week over the six-week period. At the end of the six-week intervention, the rats were sacrificed for further analysis. Plasma and liver samples were collected and stored for subsequent examinations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of FBG, blood lipids, weight, serum insulin (INS), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin (ALB)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFBG levels of all rats were measured through tail vein using the Roche Accu-Chek blood glucose meter before modeling, before intervention, and at the end of each week during the intervention period, following a 12-hour fast. The animal weight were measured before modeling, before and after intervention. The serum was separated through centrifugation at 3000 r/min, at 4℃ for 15 min, and serum concentrations of TC, TG, LDL, HDL, AST, ALT and ALB were determined by enzyme colorimetry. Insulin levels were measured by radioimmunoassay. Homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity (HOMA-ISI) were computed with the following formula: HOMA-IR = (Fasting glucose (mmol/L) \u0026times; Fasting serum insulin (\u0026micro;U/mL))/22.5. HOMA-ISI\u0026thinsp;=\u0026thinsp;22.5 / {Fasting glucose (mmol/L) \u0026times; fasting serum insulin (\u0026micro;U/mL)}.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin (HE) staining\u003c/h2\u003e \u003cp\u003eHematoxylin and eosin (H\u0026amp;E) staining was performed to examine liver morphology. Livers were collected in 4% paraformaldehyde solution for 24 h, and embedded in paraffin wax blocks after dehydrating through a serial alcohol gradient. All sections were cut at 5 \u0026micro;m sections through a microtome, and processed for staining with H\u0026amp;E. After staining, the sections were dehydrated with ascending concentrations of ethanol and xylene. Images were captured using an optical microscope (BX53; Olympus Optical).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOil Red O staining (G1261)\u003c/h2\u003e \u003cp\u003eOil Red O staining was used to evaluate liver steatosis. Mix Oil Red O stain A and B with the radio of 3:2 and place for 10 min to form modified Oil Red O stain solution. 10-\u0026micro;m-thick frozen liver sections were washed by water after fix in 10% formalin for 10 min and soaked in 60% isopropanol for 30s. Staining was performed in modified Oil Red O stain solution for 10min. The tissue slices were placed in 60% isopropanol to continue colour separation. After staining, the sections were staining by Mayer\u0026rsquo;s Hematoxylin solution for 1 min and rinsed by tap water for 10 min. Images were captured using an optical microscope (BX53; Olympus Optical).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMasson staining (G1346)\u003c/h2\u003e \u003cp\u003eMasson staining was used to evaluate liver fibrosis. 5-\u0026micro;m-thick paraffin sections were routinely dewaxed to distilled water and incubated in Mordant Solution (12h), Celestite Blue Solution (2min), Mayer hematoxylin (2min). After differentiation with Acid Differentiation Solution, the sections were washed with distilled water to stop differentiation, and then treated with Ponceau-Acid Fuchsin Solution (10min), Phosphomolybdic Acid Solution (10min), Aniline Blue Solution (5min). Images were captured using an optical microscope (BX53; Olympus Optical).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eThe liver paraffin sections underwent dewaxing to water and were subsequently subjected to heating in sodium citrate antigen retrieval solution in a microwave oven for 15 minutes. Following PBS washing, goat serum was applied to block the sections for 10 minutes. A specific primary antibody (listed in Supplementary Table\u0026nbsp;1) was then applied for overnight incubation at 4 ℃, succeeded by incubation with immunohistochemical secondary antibodies for 10 minutes at room temperature. After a 3-minute incubation with DAB solution, all sections were examined using an optical microscope (Nikon 80i, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe liver paraffin sections were dewaxed to water and heated in sodium citrate antigen retrieval solution in a microwave oven for 15 minutes. Subsequent to PBS washing, the sections were blocked with immunostaining blocking solution for 1 hour. Overnight incubation with a specific primary antibody (listed in Supplementary Table\u0026nbsp;1) at 4 ℃ was followed by incubation with fluorescent secondary antibodies for 2 hours at room temperature in the dark. After a 5-minute incubation with DAPI, all sections were observed via fluorescent microscopy (Nikon 80i, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eLiver tissue was lysed using RIPA lysis buffer containing protein phosphatase inhibitor, followed by centrifugation at 12,000\u0026times;g, 4℃ for 10 min, and the supernatant was collected. Same amount of protein sample was separated by SDS\u0026ndash;polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk solution for 2 hours, then incubated with primary antibodies (listed in Supplementary Table\u0026nbsp;2) at 4℃ overnight. After thrice washing for 10 minutes with TBST, the membranes were incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:10000) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (=\u0026thinsp;1:20000) for 1.5 hours at room temperature. The target proteins were visualized using an enhanced chemiluminescence substrate and chemiluminescence imaging system. Band intensities were detected and analyzed using image J software, Version 1.8.0.112 (National Institutes of Health (NIH)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR ( qPCR)\u003c/h2\u003e \u003cp\u003eThe extraction of total RNA utilized the HiPure Total RNA Mini Kit, followed by reverse transcription using the Reveaid First Strand cDNA Synthesis Kit. For qPCR analysis, 2 \u0026micro;l of the resulting cDNA underwent amplification and measurement employing the Power Sybr Green PCR Master Mix. Reactions were executed on a Bio-Rad CFX Maestro 1.0 ABI PRISM 7300 real-time cycler (Applied Biosystems, Foster City, CA). The protocol included a melting step at 95\u0026deg;C for 10 minutes, followed by 49 amplification cycles (95\u0026deg;C, 10 s; 55\u0026deg;C, 30 s; 65\u0026deg;C, 5 s). mRNA levels for each gene were determined utilizing the ΔΔCT method with GAPDH serving as a reference. The primers utilized for RT-PCR are detailed in Table\u0026nbsp;4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscope\u003c/h2\u003e \u003cp\u003eLiver tissue specimens were meticulously processed to ensure accurate characterization of microstructural alterations underlying metabolic associated fatty liver disease (MAFLD) pathogenesis. Initially, liver samples were meticulously fixed in 2.5% glutaraldehyde solution. Subsequently, a systematic dehydration process was employed utilizing a graded series of ethanol concentrations ranging from 30\u0026ndash;100%, facilitating the removal of water from the tissue while maintaining structural integrity. Following dehydration, the specimens underwent critical point drying to further eliminate residual moisture. To enhance electron conductivity and imaging quality, the dehydrated liver samples were coated with a thin layer of platinum via sputtering. Ultrastructural observations of liver microarchitecture were performed utilizing a transmission electron microscope (JEM-1400, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eQuantitative data were presented as mean values accompanied by standard error of mean (SEM). Statistical comparisons were conducted employing rigorous analytical methods, including one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-hoc test to determine intergroup differences. Significance levels were set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ensuring robust statistical evaluation and interpretation of experimental findings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eInfluence of EA on glycolipid metabolism and insulin resistance in T2DM model rats\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B depicted significant weight and FBG level increases in T2DM rats before intervention, confirming successful T2DM model establishment. Throughout the study, FBG levels in the T2DM group continued to rise, significantly peaking post-intervention. Compared to pre-intervention levels, FBG concentrations in EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met groups notably decreased after the fourth, fifth, and sixth weeks of treatment. However, the FBG levels in the EA\u0026thinsp;+\u0026thinsp;Met group, although the lowest, did not show statistical significance compared to the other groups. Conversely, FBG levels in the EA\u0026thinsp;+\u0026thinsp;Compd C group were markedly higher than those in the EA group post-intervention. FBG levels in the Con group remained within the normal range throughout the study. Following intervention, T2DM rats exhibited insulin resistance and lipid metabolism disturbances. Serum TC, TG, LDL, weight, and HOMA-IR levels significantly surpassed those of the Con group, while serum HDL levels and HOMA-ISI notably decreased in the T2DM group. Conversely, all treatment groups showed significant reductions in TC, TG, LDL, weight, and HOMA-IR levels, with notable increases in HDL levels and HOMA-ISI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT/PKB) signaling pathway / glucose transporter-4 (GLUT4) signaling pathway, crucial for insulin-stimulated glucose transport, showed suppression in the T2DM group compared to the Con group in the results of Western Blotting. However, EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met treatments reversed the downregulation of hepatic PI3K, AKT, and GLUT4 protein expression. While the EA\u0026thinsp;+\u0026thinsp;Met group exhibited greater increases in protein expression, the differences were not statistically significant. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAMPK, a key regulator of fatty acid, glucose homeostasis, and insulin sensitivity, was investigated along with its downstream signaling pathways PPARα/CPT1A, SREBP1c and PGC-1α/PCK2, which responsible for gluconeogenesis, fat generation, and fatty acids oxidative utilization. HFD\u0026thinsp;+\u0026thinsp;STZ treatment led to alterations in protein expression levels, which were reversed by EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met treatments. qPCR results further confirmed these findings, showing significant changes in mRNA levels of related genes. The results showed that PPARα and CPT1A protein expression were distinctly suppressed, while PGC-1α and PCK2 protein expression were distinctly upgraded by HFD\u0026thinsp;+\u0026thinsp;STZ treatment as compared to the Con group. However, EA, Met and EA\u0026thinsp;+\u0026thinsp;Met treatment reversed the changes of PPARα, CPT1A, PGC-1α and PCK2 protein expression in T2DM rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). The results of qPCR further confirmed that the mRNA levels of PGC-1α and SREBP-1c were significantly increased in T2DM group and decreased by EA, Met and EA\u0026thinsp;+\u0026thinsp;Met treatment, meanwhile the mRNA levels of PPARα were decreased in T2DM group and increased in EA, Met and EA\u0026thinsp;+\u0026thinsp;Met groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further performed intraperitoneal injections of the AMPK inhibitor (Compound C) in T2DM rats treated with EA, to investigate the target of EA in the treatment of T2DM. The results revealed increased levels of FBG, TC, TG, LDL, HOMA-IR, and decreased HDL and HOMA-ISI compared with the EA group. Additionally, the expression of PI3K/AKT/GLUT4, PPARα/CPT1A signaling pathways was down-regulated, while PGC-1α/PCK2 signaling pathway and SREBP1c expression was upgraded in the EA\u0026thinsp;+\u0026thinsp;Compd C group compared to the EA group.\u003c/p\u003e \u003cp\u003eEA-mediated autophagy ameliorated hepatic function and morphological change in T2DM rats.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, histopathological analysis (H\u0026amp;E staining) revealed evident liver damage in T2DM rats, characterized by hepatocyte swelling, lipid vacuoles, ballooning, inflammation, and lobular structure irregularities. Notably, after 6 weeks of EA and Met treatment, these pathological features improved significantly, with EA\u0026thinsp;+\u0026thinsp;Met nearly restoring hepatocyte morphology to normal levels, indicating a potent synergistic effect of the two interventions. Furthermore, staining with Oil Red O (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), a dye commonly used to visualize lipid droplets, demonstrated substantial lipid accumulation in T2DM rat livers compared to the Con group, which decreased significantly after EA and Met treatment, suggesting an attenuation of hepatic steatosis\u0026mdash;a prevalent complication in T2DM-associated liver dysfunction. The combined group exhibited lower lipid accumulation than the EA or Met groups. Furthermore, serum AST, ALT, and ALB levels, the critical hepatic function indicators, were used to appraise the hepatoprotective effect of EA, Met and EA\u0026thinsp;+\u0026thinsp;Met in T2DM rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In rats with T2DM, serum AST and ALT levels exhibited a decline, concomitant with an elevation in serum ALB levels. Upon administering three distinct treatments (EA, Met, EA\u0026thinsp;+\u0026thinsp;Met), the trend in AST and ALT levels showed a obvious decrease, whereas ALB levels experienced a subsequent increase. Remarkably, the combined approach of EA alongside medication demonstrated superior efficacy in modulating ALT levels compared to both metformin monotherapy and EA interventions. This finding not only underscored the potential synergistic effects of combining therapeutic modalities but also highlighted EA's promising role in ameliorating hepatic function in the context of T2DM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe role of autophagy in T2DM-induced MAFLD emerges as pivotal, serving as a cellular mechanism that mitigates liver steatosis and maintains cellular homeostasis. Transmission electron microscopy revealed pronounced differences between the control and T2DM groups in liver cell composition. In the T2DM group, substantial lipid droplets were evident alongside blurred mitochondrial structure, expanded endoplasmic reticulum, and diminished autophagosomes, whereas EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met groups showed restored cellular morphology with increased autophagosomes and reduced lipid droplets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The autophagy activity in liver of T2DM rats was examined by western blotting, qPCR, immunohistochemistry and immunofluorescence. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C and F-G, a notable downward trend in both protein and mRNA expression of LC3II was observed in the T2DM group, indicating suppressed autophagy activity. Conversely, the EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met groups exhibited a significant upsurge in LC3II expression, suggesting that EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met interventions partially restored autophagy activity impaired by T2DM. Additionally, to delve deeper into EA's mechanism of action in combating diabetes by ameliorating hepatic lipid accumulation through autophagy, we scrutinized the effects of the AMPK/mTOR pathway in the livers of T2DM rats. The results from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E and G indicated that HFD\u0026thinsp;+\u0026thinsp;STZ treatment significantly increased p-mTOR/mTOR and mTOR mRNA levels, while decreasing p-AMPK/AMPK and AMPK mRNA levels. Conversely, EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met treatment restored these levels. This suggests that EA may regulate liver autophagy activity through the AMPK/mTOR pathway. Immunohistochemical analysis mirrored the western blotting and qPCR findings for AMPK and mTOR expression in the liver. Notably, the EA\u0026thinsp;+\u0026thinsp;Met group exhibited notably superior hepatic AMPK expression compared to the EA and Met groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe design of the combination of EA and AMPK inhibitor group further validated the above conclusion. This study also showed that Compound C effectively inhibited the promotion of autophagy by EA in liver, including a marked augmented level of mTOR and low expression of AMPK and LC3II, which could explain the increase of lipid droplet, the decrease of autophagosomes, hepatic morphological damage, and reduced liver fuction.\u003c/p\u003e \u003cp\u003eEA reduces liver fibrosis induced by TGF β1/smad3 pathway\u003c/p\u003e \u003cp\u003eSpecific stains, like Masson staining, were used to detect collagenous fibers and evaluate hepatic fibrosis. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, the Masson staining revealed a stark contrast between the normal and model groups in terms of hepatic fibrosis, with the model group displaying a significant increase in collagen fibers. Conversely, treatment with EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met notably reversed the presence of blue collagen fibers, particularly notable in the EA\u0026thinsp;+\u0026thinsp;Met group. These findings indicated that EA effectively mitigated T2DM-induced hepatic fibrosis. Transmission electron microscopy revealed significant collagen fibers in the perisinusoidal space of the liver in the T2DM group, a feature notably reduced in the EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). The immunofluorescence results had showed that the model group exhibited increased α-SMA expression compared to the Con group. However, in the EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met groups, α-SMA expression decreased significantly compared to the model group, indicating three treatment\u0026rsquo;s potential to inhibit hepatic fibrosis. This was further supported by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C, F-G).\u003c/p\u003e \u003cp\u003eThe association of the TGFβ1/Smad3 pathway with liver fibrosis has been extensively demonstrated. To investigate whether the protective effect of EA involves inhibition of the TGFβ1/Smad3 pathway, we assessed changes in this pathway using Western blotting, immunohistochemistry, and qPCR methods. Primarily, Western blot and immunohistochemistry analyses revealed significantly elevated expressions of TGFβ1 and Smad3 in the T2DM group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, and C). However, after 6 weeks of EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met treatment, these expressions were notably suppressed. Furthermore, qPCR results mirrored these findings: liver tissue from T2DM rats exhibited increased mRNA expression of TGFβ1, Smad2, and Smad3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Yet, treatment with EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met effectively inhibited the mRNA expression of TGFβ1, Smad2, and Smad3. To further identify the involvement of glycolipid metabolism and autophagy mediated by AMPK in the formation of liver fibrosis, the therapeutic action of EA was further evaluated using intraperitoneal injection of compound C. The results indicated that the expression of TGFβ1, smad2/3, and α-SMA were significantly increased after EA\u0026thinsp;+\u0026thinsp;Compd C treatment compared with the EA group. Masson staining and transmission electron microscopy showed that the EA\u0026thinsp;+\u0026thinsp;Compd C group exhibited greater degree of collagenous fiber compared with the EA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C, E, G).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIndividuals diagnosed with T2DM are susceptible to hepatocyte injury and fibrosis, even when presenting with isolated steatosis in the initial stages. This progression underscores the significance of managing T2DM-induced hepatic steatosis to forestall the development of fibrosis and the onset of MAFLD. The presence of liver fibrosis, often a precursor to cirrhosis, is notably more prevalent among individuals with diabetes, highlighting the urgent need for effective therapeutic interventions (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). However, the current clinical landscape is marked by a dearth of FDA-approved drugs specifically tailored for treating MAFLD (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Addressing this gap, the present study delves into the protective potential of EA against T2DM-induced hepatic steatosis and ensuing fibrosis. Utilizing a T2DM rat model, this investigation scrutinizes the comparative efficacy of EA, metformin, and a combination thereof in mitigating the progression of hepatic complications. Metformin, a cornerstone medication in T2DM management, operates through the activation of the AMPK pathway, thereby enhancing liver function and insulin sensitivity (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The study endeavored to elucidate whether the combined administration of EA and metformin yields synergistic effects, potentially offering a novel therapeutic approach for ameliorating hepatic dysfunction in T2DM. Our studies indicated that hyperglycemia, hyperlipidemia and IR induced by high-fat diet (HFD) and injection of STZ promoted lipid accumulation, pathological and functional damage, and resulting in hepatic steatosis and fibrosis. Meanwhile, we found that EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met enhanced the activation of AMPK, which in turn promoted the expression of PPARα/CPT1A pathway, inhibited the expression of SREBP1c and PGC-1α/PCK2 pathway, futher improved levels of autophagy mediated by AMPK/mTOR pathway and thus suppressed TGFβ1/Smad2/3 signaling pathway, ultimately exerting its effect on ameliorating hepatic steatosis and fibrosis in T2DM rats. In a nutshell, our study revealed that the three treatment prominently alleviated T2DM-induced MAFLD, the mechanism was likely associated with the regulation of glycolipid metabolism and autophagy mediated by AMPK signaling pathway in liver.\u003c/p\u003e \u003cp\u003eThe interplay between T2DM and MAFLD entails intricate bidirectional regulation mechanisms, influencing each other's progression and severity (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). T2DM and IR instigate hepatic triglyceride accumulation, exacerbating MAFLD, while lipid toxicity and hyperglycemia further fuel the development of MAFLD (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Conversely, the presence of MAFLD aggravates insulin resistance, establishing a detrimental cycle of mutual reinforcement. The AMPK pathway emerges as a pivotal player in bridging the pathophysiological connection between T2DM and MAFLD, orchestrating glycolipid metabolism and insulin resistance (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In our study using T2DM rats induced by HFD\u0026thinsp;+\u0026thinsp;STZ treatment, we observed that intervention with EA treatment in T2DM rodent models exhibited hepatoprotective effects by bolstering AMPK activity. Numerous studies emphasized AMPK's pivotal role in T2DM and MAFLD development and progression (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). AMPK, comprising one catalytic subunit (α1 or α2) and two regulatory subunits, serves as a vital regulator of energy balance in liver, muscle, and adipose tissues (\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). AMPK conserves ATP and modulates energy metabolism by activating catabolic pathways and inhibiting anabolic pathways, including fatty acid oxidation, hepatic lipogenesis, glucose uptake and output, and insulin sensitivity regulation (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). AMPK phosphorylation inhibits transcription factors that induce gluconeogenesis and lipogenic programs, notably PCK2 and SREBP1, reducing liver glucose and fat accumulation (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Additionally, AMPK promotes fatty acid entry into mitochondria and oxidation through the CPT1 system via PPARα (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Regular exercise enhanced whole-body insulin sensitivity by activating AMPK, as evidenced by epidemiological data indicating lower prevalence of metabolic syndrome diseases among physically active individuals (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Strategies aimed at activating AMPK hold significant promise for both the prevention and treatment of T2DM and MAFLD. To delve deeper into the mechanism underlying EA, we conducted experiments utilizing T2DM rat models subjected to EA treatment, assessing the expression of molecular markers associated with AMPK. Throughout our rigorous investigation, stark disparities were observed between the control group and the T2DM rats. Specifically, levels of FBG, serum lipid components (including TG, TC, LDL), and HOMA-IR significantly escalated in the T2DM group. Conversely, serum HDL and HOMA-ISI levels notably declined. These findings underscored the manifestation of evident IR and dysregulation in glycolipid metabolism within the T2DM rat models. Following a 6-week intervention period, all three treatment modalities not only induced a substantial decrease in FBG levels from the fourth to sixth week but also precipitated a remarkable reduction in TC, TG, LDL, and HOMA-IR levels, coupled with a significant elevation in HDL and HOMA-ISI levels. These salutary effects were consonant with the activation of AMPK, culminating in the upregulation of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), and glucose transporter type 4 (GLUT4), alongside the inhibition of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), phosphoenolpyruvate carboxykinase 2 (PCK2), and sterol regulatory element-binding protein 1c (SREBP1c). Consequently, these cascades led to diminished adipogenesis and gluconeogenesis, augmented fatty acid oxidation, and heightened insulin sensitivity, thereby substantiating the therapeutic potential of AMPK activation in ameliorating the metabolic derangements associated with T2DM and MAFLD. The regulatory impact of EA, when combined with Met intervention, on the expression of hepatic AMPK surpassed that of both EA and Met administration. This superiority underscored the potential advantage of integrating EA with Met therapy in clinical practice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the impact of enhanced glycolipid metabolism on the liver, we examined liver function and pathological changes. Our observations revealed elevated levels of ALT and AST, alongside reduced ALB levels in T2DM rats compared to the control group. Notably, the livers of T2DM rats exhibited inflammation, lipid vacuoles, ballooning, and significant fat accumulation, indicating liver metabolic dysfunction and hepatocyte steatosis. Furthermore, our results indicated that the three treatments effectively alleviated liver injury induced by IR. This was evidenced by decreased AST and ALT levels, increased ALB levels in serum, and improvements in hepatic morphology, along with reduced areas of positive Oil Red O staining. Moreover, electron microscope analysis revealed a notable presence of autophagosomes in liver tissue across the three treatment groups.\u003c/p\u003e \u003cp\u003eAutophagy, an essential system for maintaining cellular homeostasis, mitigates liver injury, oxidative stress, and inflammation by breaking down and recycling misfolded proteins and damaged organelles resulting from lipid peroxidation. This process thereby prevents the onset of hepatic steatosis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Lipophagy, a subtype of autophagy, targets intracellular lipid droplets (LDs) for degradation, aiming to regulate liver fat stores (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Research indicated that compromised autophagic flux contributed to lipid droplet accumulation in hepatocytes, exacerbating hepatic steatosis and damage in T2DM rats. LC3-II, a protein crucial for autophagosome formation and maturation, serves as a reliable marker for assessing autophagic activity (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). In the present study, both the protein and mRNA expression levels of LC3-II were significantly reduced in the T2DM group, suggesting suppressed autophagic activation. This finding correlated with the observed increase in hepatic lipid deposition and morphological alterations. Conversely, LC3B-II levels were markedly elevated, accompanied by a reduction in LDs following the three treatments compared to the T2DM group. This suggested that enhanced autophagy facilitated lipid clearance in the liver. Consequently, EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met treatments may alleviate hepatic lipid accumulation by inducing autophagy in T2DM. Central to the regulation of autophagy in MAFLD is the AMPK /mTOR pathway, which has been extensively documented (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). AMPK, a highly conserved serine/threonine-protein kinase across evolutionary scales, serves as a pivotal energy sensor, thereby regulating metabolic homeostasis and amplifying autophagy (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Conversely, mammalian target of rapamycin (mTOR), a central regulator of cell growth, inhibits autophagy by integrating nutrient signals and growth factors. mTORC1, a complex of mTOR (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e), phosphorylates ULK1 at Ser757, impeding its interaction with AMPK, thus halting autophagy. AMPK, as the upstream regulator of mTOR, negatively modulates mTOR activation either directly by phosphorylating mTORC1 or by phosphorylating various components of mTORC1. Moreover, AMPK fosters autophagy by directly activating ULK1 through phosphorylation at Ser555 and Ser777, facilitating the release of ULK1 from mTORC1. Consequently, the activated ULK1 kinase complex recruits other autophagy proteins, instigating autophagy induction (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan additionalcitationids=\"CR66 CR67\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). However, in conditions such as steatosis and hypernutrition, autophagy initiation is hindered due to alterations in AMPK and mTOR signaling. The current study revealed a significant decrease in the p-AMPK/AMPK ratio and AMPK mRNA alongside a remarkable increase in the p-mTOR/mTOR ratio and mTOR mRNA. This was coupled with the inhibition of autophagy, as evidenced by the notable decrease in LC3-II levels in the T2DM group. However, autophagy was restored in the groups treated with EA, Met, and EA\u0026thinsp;+\u0026thinsp;Met, achieved through elevated AMPK expression and reduced mTOR expression levels, consequently leading to increased LC3-II levels. Moreover, our results showed that EA-induced autophagy and improvement of hepatic lipid droplet accumulation and glycolipid metabolism was partly disrupted by Compound C, an inhibitor of AMPK, in the EA\u0026thinsp;+\u0026thinsp;Compd C group. Taken together, these results suggest that the beneficial effects of EA may stem from enhanced autophagy via the AMPK/mTOR pathway.\u003c/p\u003e \u003cp\u003eChronic liver injury caused by T2DM and metabolic disorders gradually progresses to fibrosis, which is characterized by excess deposition of collagen between hepatocytes and hepatic sinusoids to repair the liver damage (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Collagen, a pivotal structural protein within the extracellular matrix (ECM), serves as a fundamental component in maintaining tissue integrity and function. In the liver, hepatic stellate cells (HSCs) emerge as the primary source of collagen synthesis, playing a critical role in the dynamic equilibrium of ECM turnover and regulation. In response to liver injury, HSCs undergo a phenotypic transformation into activated myofibroblast-like cells (MFCs), which are proficient in ECM production, notably collagen types I and III, and formate α-smooth muscle actin (α-SMA) stress fibers (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). This transition marks a pivotal event in the pathogenesis of liver fibrosis, characterized by excessive ECM deposition and aberrant tissue remodeling. Central to the fibrotic cascade is the multifaceted cytokine transforming growth factor-beta (TGF-β), which orchestrates various cellular processes, including HSCs activation and proliferation. Within the liver microenvironment, TGF-β1 emerges as a key isoform, synthesized by both liver parenchymal cells and activated HSCs, exerting its effects through a Smad3-dependent signaling pathway (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). Upon binding to the TGF-β II receptor on the cell membrane of HSCs, TGF-β1 initiates a signaling cascade culminating in the activation of intracellular mediators, notably Smad proteins (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). Subsequently, the Smad complex, comprising Smad2, Smad3, and Smad4, translocates into the nucleus, where it modulates gene transcription, including the upregulation of collagen expression. By directly binding to the collagen promoter region, the Smad complex exerts transcriptional control, thereby fostering ECM production and deposition, thereby perpetuating the fibrotic response in the liver (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). Understanding the intricate interplay between HSCs, TGF-β signaling, and collagen synthesis offers crucial insights into the molecular mechanisms underpinning liver fibrosis. Targeting these pathways holds promise for the development of novel therapeutic strategies aimed at mitigating fibrotic progression and restoring hepatic homeostasis. Our results showed that the expressions of α-SMA, a fibroblast marker, were abundantly increased in the T2DM group. The pathological characteristics of T2DM in massson staining and electron-microscope exhibited an increase in hepatic fibrosis, indicating activation of HSCs. We also found that HFD\u0026thinsp;+\u0026thinsp;STZ treatment distinctly increased the expressions of TGFβ1, smad2, and smad3. These data prompted that T2DM-induced aberrant glycolipid metabolism possess a critical role in liver injury and fibrosis in T2DM rats. It is worth mentioning that the three treatments could attenuated this effect and significantly ameliorate hepatic fibrosis and decrease the expression of TGFβ1, smad2, and smad3 in T2DM rats, suggesting that the antifibrotic activity of three treatments may be associated, at least partially, with TGFβ1/smad2/3 signaling pathway activity attenuation.\u003c/p\u003e \u003cp\u003eHowever, there arised a question: was the upregulation of autophagy and glycolipid metabolism by EA linked to the downregulation of TGFβ1/SMAD3 signaling, or was it an independent molecular event requiring further investigation? As previously mentioned, AMPK-mediated glycolipid metabolism and autophagy contributed to restoring liver structure and function, thereby preventing liver fibrosis by mitigating factors causing liver damage. Rangnath Mishra investigated the interplay between AMPK and the TGFβ1/SMAD3 pathway. Their study revealed that pharmacologically activating AMPK inhibited TGFβ-induced secretion of collagen types I and IV and fibronectin, driven by Smad3-binding cis-elements. In our present study, as anticipated, the introduction of Compound C, an AMPK inhibitor, suppressed AMPK activation and partially reversed the beneficial effects of EA on the TGFβ1/SMAD3 signaling pathway and liver fibrosis. This finding confirmed a direct relationship between AMPK activation and the anti-fibrotic effects of EA treatment. Considering the aforementioned studies, the AMPK signaling pathway mediatedEA's beneficial effects in delaying the progression of MAFLD in T2DM rats.\u003c/p\u003e \u003cp\u003eIn conclusion, to the best of our knowledge, the results of the present study showed that EA played a critical role in ameliorating in MAFLD in T2DM rats. Specifically, EA intervention improved FBG, serum lipids, insulin sensitivity and hepatic function, and inhabited liver steatosis and fibrosis of T2DM rats. Furthermore, we demonstrated insights that EA treatment was likely through the mechanisms of the up-regulating of hepatic AMPK signaling pathway, which mediated glycolipid metabolism and autophagy, inhibited TGFβ1/SMAD3 pathway-induced fibrosis. Moreover, the above effects of EA were consistent with metformin. The combination of EA and metformin had significant advantages in increasing hepatic AMPK expression, improving liver morphology, lipid droplet infiltration, fibrosis, and reducing serum ALT levels. Taken together, our findings indicated that EA might be taken as an effective therapeutic strategy and improving the efficacy of metformin in MAFLD. Therefore, the combined approach of EA and Met in clinical practice offers a personalized and holistic treatment strategy that considers the individual's constitutional differences and the dynamic nature of metabolic disorders. Through synergistic interactions, acupuncture may enhance the efficacy of pharmacotherapy while minimizing adverse effects and promoting overall well-being. Future research endeavors should focus on elucidating the underlying mechanisms responsible for the superior regulatory effect of EA combined with Met on liver AMPK expression. Additionally, multiple animal studies and larger clinical trials are warranted to validate the therapeutic efficacy and safety of this integrated approach in diverse patient populations with metabolic disorders. Ultimately, integrating EA with Met holds promise for advancing precision medicine and improving patient outcomes in the management of metabolic diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eHD: Conceptualization, Writing\u0026mdash;original draft, Project administration.\u0026nbsp;SS: Data curation, Methodology. RL: Supervision, Funding acquistion.\u0026nbsp;SH: Writing \u0026ndash; review \u0026amp; editing. SZ: Visualization.\u0026nbsp;SL: Software.\u0026nbsp;XL:\u0026nbsp;Formal analysis.\u0026nbsp;WG:\u0026nbsp;Validation.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the following grants: 1. National Natural Science Foundation of China (Grant Nos. 81973935); 2. Beijing Municipal Natural Science Foundation (Grant Nos. 7232276).\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors thank the School of Acupuncture - Moxibustion, and Tuina, Beijing University of Chinese Medicine for supplied experimental equipment and research environment.\u003c/p\u003e\n\u003cp\u003eEthical Approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe protocol received approval from the Laboratory Animal Welfare and Ethics Committee of Beijing University of Chinese Medicine (BUCM-4-2021051301-2026).\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research has\u0026nbsp;no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003eData availability\u0026nbsp;statement\u003c/p\u003e\n\u003cp\u003eThe study\u0026apos;s original contributions are available in the Supplementary Material. For additional information, please contact the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMuthiah MD, Cheng Han N, Sanyal AJ. A clinical overview of non-alcoholic fatty liver disease: A guide to diagnosis, the clinical features, and complications-What the non-specialist needs to know. Diabetes, obesity \u0026amp; metabolism. 2022;24 Suppl 2:3-14.\u003c/li\u003e\n\u003cli\u003eAhmad E, Lim S, Lamptey R, Webb DR, Davies MJ. Type 2 diabetes. Lancet (London, England). 2022;400(10365):1803-20.\u003c/li\u003e\n\u003cli\u003eXie J, Huang H, Liu Z, Li Y, Yu C, Xu L, et al. The associations between modifiable risk factors and nonalcoholic fatty liver disease: A comprehensive Mendelian randomization study. Hepatology (Baltimore, Md). 2023;77(3):949-64.\u003c/li\u003e\n\u003cli\u003eButt AS, Hamid S, Haider Z, Sharif F, Salih M, Awan S, et al. 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Journal of gastroenterology and hepatology. 2019;34(1):263-76.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"diabetology-and-metabolic-syndrome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dims","sideBox":"Learn more about [Diabetology \u0026 Metabolic Syndrome](http://dmsjournal.biomedcentral.com/)","snPcode":"13098","submissionUrl":"https://submission.nature.com/new-submission/13098/3","title":"Diabetology \u0026 Metabolic Syndrome","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"T2DM, MAFLD, EA, AMPK signaling pathway, autophagy.","lastPublishedDoi":"10.21203/rs.3.rs-4475748/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4475748/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eRecent studies havehighlighted type 2 diabetes (T2DM) as a significant risk factor for the development of metabolic dysfunction-associated fatty liver disease (MAFLD). This investigation aimed to assess electroacupuncture's (EA) impact on liver morphology and function in T2DM rats, furnishing experimental substantiation for its potential to stall MAFLD progression in T2DM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e T2DM rats were induced by a high-fat diet and a single intraperitoneal injection of streptozotocin, and then randomly assigned to five groups: the T2DM group, the electroacupuncture group, the metformin group, combination group of electroacupuncture and metformin, combination group of electroacupuncture and Compound C. The control group received a standard diet alongside intraperitoneal citric acid - sodium citrate solution injections. After a 6-week intervention, the effects of each group on fasting blood glucose, lipids, liver function, morphology, lipid droplet infiltration, and fibrosis were evaluated. Techniques including Western blotting, qPCR, immunohistochemistry, and immunofluorescence were employed to gauge the expression of key molecules in AMPK-associated glycolipid metabolism, insulin signaling, autophagy, and fibrosis pathways. Additionally, transmission electron microscopy facilitated the observation of liver autophagy, lipid droplets, and fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Our studies indicated that hyperglycemia, hyperlipidemia and IR promoted lipid accumulation, pathological and functional damage, and resulting in hepatic steatosis and fibrosis. Meanwhile, EA enhanced the activation of AMPK, which in turn improved glycolipid metabolism and autophagy through promoting the expression of PPARα/CPT1A and AMPK/mTOR pathway, inhibiting the expression of SREBP1c, PGC-1α/PCK2 and TGFβ1/Smad2/3 signaling pathway, ultimately exerting its effect on ameliorating hepatic steatosis and fibrosis in T2DM rats. The above effects of EA were consistent with metformin. The combination of EA and metformin had significant advantages in increasing hepatic AMPK expression, improving liver morphology, lipid droplet infiltration, fibrosis, and reducing serum ALT levels. In addition, the ameliorating effects of EA on the progression of MAFLD in T2DM rats were partly disrupted by Compound C, an inhibitor of AMPK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e EA upregulated hepatic AMPK expression, curtailing gluconeogenesis and lipogenesis while boosting fatty acid oxidation and autophagy levels. Consequently, it mitigated blood glucose, lipids, and insulin resistance in T2DM rats, thus impeding liver steatosis and fibrosis progression and retarding MAFLD advancement.\u003c/p\u003e","manuscriptTitle":"Strategy for treating MAFLD: Electroacupuncture alleviates hepatic steatosis and fibrosis by enhancing AMPK mediated glycolipid metabolism and autophagy in T2DM rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 14:06:55","doi":"10.21203/rs.3.rs-4475748/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-15T14:38:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-15T14:24:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-15T04:52:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125229736587898943575555991617610015968","date":"2024-06-08T11:58:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269163770086812243502606505807490845534","date":"2024-06-05T04:08:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-29T20:16:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-29T20:15:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-28T06:30:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Diabetology \u0026 Metabolic Syndrome","date":"2024-05-25T07:53:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"diabetology-and-metabolic-syndrome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dims","sideBox":"Learn more about [Diabetology \u0026 Metabolic Syndrome](http://dmsjournal.biomedcentral.com/)","snPcode":"13098","submissionUrl":"https://submission.nature.com/new-submission/13098/3","title":"Diabetology \u0026 Metabolic Syndrome","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4bd0d83-2581-4e24-b690-e3f5404a0fad","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:03:02+00:00","versionOfRecord":{"articleIdentity":"rs-4475748","link":"https://doi.org/10.1186/s13098-024-01432-7","journal":{"identity":"diabetology-and-metabolic-syndrome","isVorOnly":false,"title":"Diabetology \u0026 Metabolic Syndrome"},"publishedOn":"2024-09-11 15:57:45","publishedOnDateReadable":"September 11th, 2024"},"versionCreatedAt":"2024-06-10 14:06:55","video":"","vorDoi":"10.1186/s13098-024-01432-7","vorDoiUrl":"https://doi.org/10.1186/s13098-024-01432-7","workflowStages":[]},"version":"v1","identity":"rs-4475748","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4475748","identity":"rs-4475748","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}