Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic condition characterized by disrupted lipid metabolism and inflammation in the liver(Puri and Sanyal 2012). It is a significant concern because of its potential for progression to more severe conditions, including non-alcoholic steatohepatitis NASH, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC)(McPherson, Hardy et al. 2015, Geh, Anstee and Reeves 2021). Emerging research has underscored the critical role of the gut-liver axis in the development of NAFLD, with multiple factors implicated in its progression(Song and Zhang 2022). Among these factors, Trimethylamine N-Oxide (TMAO), a metabolite derived from dietary sources, such as fish, red meat, and eggs, has gained attention for its inflammation-inducing properties(Ufnal, Zadlo and Ostaszewski 2015). TMAO has been linked to the upregulation of proinflammatory cytokines, including Tumor Necrosis Factor-alpha (TNFα) and Interleukin-6 (IL-6), which contribute to the inflammatory responses(Gao, Liu et al. 2014). Furthermore, TMAO has been associated with NAFLD progression by affecting lipid metabolism(He, Tan et al. 2020), promoting apoptosis(Yang and Zhang 2021), and inducing profibrotic factors(Fang, Zheng et al. 2021). One key signaling pathway serving TMAO to induce inflammation is the P38 mitogen-activated protein kinase (P38MAPK) pathway(Yang, Lin et al. 2019). Inflammation is a key feature of NAFLD, and P38MAPK contributes to this inflammatory response through several mechanisms(Deng, Tang et al. 2018). Firstly, P38MAPK is involved in the activation of pro-inflammatory signaling pathways in liver cells, particularly hepatocytes and Kupffer cells(Scott and Billiar 2008, Imarisio, Alchera et al. 2017). When the liver becomes overloaded with fat, cellular stress and damage occur, leading to the release of inflammatory cytokines and chemokines(Stojsavljević, Palčić et al. 2014, Niederreiter and Tilg 2018). P38MAPK is activated in response to stress and, in turn, activates downstream transcription factors such as NF-κB(Deng, Tang et al. 2018), which promotes the expression of pro-inflammatory cytokines such as TNF-α IL-6(Thirunavukkarasu, Watkins and Gandhi 2006, Fanaei, Mard et al. 2021), exacerbating the inflammation within the liver. Additionally, P38MAPK contributes to the recruitment and activation of immune cells, such as macrophages, into the liver tissue. These immune cells play a pivotal role in the perpetuation of inflammation in NAFLD(Zhang, Fan et al. 2019). In summary, P38MAPK plays a crucial role in the pathogenesis of NAFLD by amplifying the inflammatory response in the liver, making it a potential target for therapeutic interventions aimed at reducing inflammation and mitigating disease progression. Highly Upregulated in Liver Cancer (HULC) is a long non-coding RNA (lncRNA) implicated in various aspects of liver cancer (hepatocellular carcinoma or HCC) and other liver-related diseases(Ghafouri‐Fard, Esmaeili et al. 2020). HULC was initially identified as a significantly overexpressed lncRNA in hepatocellular carcinoma (liver cancer) tissues compared to normal liver tissues(Yu, Zheng et al. 2017, Xin, Wu et al. 2018). Although HULC is primarily known for its role in liver cancer, a study conducted by Shen et al. showed that HULC is upregulated in NAFLD(Shen, Guo et al. 2019). Cui et al. showed a disturbance in lipid metabolism in HepG2 cells induced by HULC(Cui, Xiao et al. 2015). HULC has emerged as a potential regulator of the P38MAPK pathway(Shen, Guo et al. 2019). Furthermore, recent studies have reported elevated plasma TMAO levels in NAFLD patients(Moradzad, Abdi et al. 2022, Theofilis, Vordoni and Kalaitzidis 2022). These findings suggest a possible connection between TMAO, HULC, and the P38MAPK pathway in the context of NAFLD because most of the changes induced by TMAO are through P38MAPK which HULC is the master regulator of P38MAPK. Therefore, in this study, we aimed to investigate the involvment of the signaling pathway upon TMAO exposure. To do this, we hypothesized that HULC/P38MAPK is the axis of TMAO induces inflammation and apoptosis. This study aims to investigate how TMAO induces inflammation by analyzing this axis and following related responses such as TNFα and IL-6 expression and finally examine its relation to PNPPLA3 as a profibrotic factor in HepG2 cells.

2. Material and Methods: 2.1. Cell Culture, Fatty Liver Cell Model and Treatments HepG2 cells (Pasteur Institute, Iran) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine, 100 U/ml of penicillin (Gibco), and 100 μg/ml streptomycin (Gibco) in a 5% CO2 atmosphere at 37°C. We then established a fatty liver cell model as previously described (Abdolahi, Vahabzadeh et al. 2022). Briefly, to induce fatty liver conditions, approximately two million serum-starved cells were exposed to a mixture of Palmitate and Oleate fatty acids in a 1:2 ratio, resulting in a final concentration of 1200 μM, and incubated for 24 h. To verify the accumulation of fat within the cells, we utilized Nile Red dye (0.75 μg/ml, Sigma, 7385-67-3) to stain the cells fixed with 3% paraformaldehyde for 15 minutes at room temperature. 2.2. MTT Assay: For each component that we treated HepG2 cells, including TMAO (75 μM and 300 μM), SB203589 (10 μM and 50 μM), and palmitate/oleate fatty acid (1200 μM), an MTT test was conducted(Abdolahi, Vahabzadeh et al. 2022). Briefly, HepG2 cells were seeded in a 96-well plate in triplicate using complete DMEM high glucose at a density of 1.0 × 104 cells per well and incubated for 24 h. Subsequently, the culture medium was replaced with an FBS-free medium and incubated for an additional 18 h. The following day, the cells were subjected to the specific treatment protocol mentioned earlier for 24 h. To assess cell viability in each group, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT; Sigma) was used. The optical absorption was measured at 570 nm using a Nanodrop spectrophotometer (Synergy HT; BioTek, USA). 2.3. LncRNA HULC(KD) HepG2 cell production using the CRISPR/Cas13 system lnc-RNA HULC was knocked down in HepG2 cells using the CRISPR/Cas13 system. To design guide RNAs (gRNAs), the Breaking‐Cas online tool was utilized (link), and these gRNAs were then synthesized (Pishgam Company,Iran). sgRNAs (Table1) were inserted into the PregRNA plasmid, which had been previously linearized using the BbsI restriction enzyme (Thermo Fisher Scientific). 2.4. RNA Extraction, cDNA Synthesis and RT-qPCR Total RNA was extracted from the cells using the (Parstous) according to the manufacturer instructions. The quality and quantity of the extracted RNA were assessed using a NanoDrop spectrophotometer (Synergy HT; BioTek, USA). For cDNA synthesis parstous kit was used for cDNA synthesis. Briefly, 2 μg of the extracted RNA was mixed with the reverse transcription master mix, which included a reverse transcriptase enzyme, random primers, and dNTPs. The mixture was incubated at 42-55°C, for 1 h. To measure the gene expression levels, quantitative real-time polymerase chain reaction (RT-qPCR) was performed using (Corrbet Rotor Gene 6000) and specific primer sets (Table2) targeting the genes of interest. 2.5. Apoptosis Assay HepG2 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and maintained at 37°C in a humidified atmosphere with 5% CO2. Cells were treated with TMAO concentrations (75µM and 300µM) for 24 hours before apoptosis analysis. Briefly, cells were collected and washed twice with ice-cold phosphate-buffered saline (PBS). Cells were resuspended in 1X Annexin V binding buffer and stained with Annexin V-FITC Detection Kit (Thermo Fisher, Lot#E00887-1630) and PI (BioLegend, Lot #132482) according to the manufacturer's instructions. Following a 15-minute incubation at room temperature in the dark, the samples were analyzed by flow cytometry. 2.6. Statistical Analysis: All experiments described in the Materials and Methods section were conducted in triplicate. RT-Qpcr data were analyzed using 2-ΔΔCq method(Livak and Schmittgen 2001). Shapiro-Wilk test was used to assess the normality of data distribution. Subsequently, one-way analysis of variance (ANOVA) was employed to evaluate the overall differences for each variant across the experimental groups. Post hoc tests were conducted to identify specific group differences, with a particular focus on comparisons with the control group. Tukey's Honestly Significant Difference (HSD) test was chosen as the post hoc analysis. Graphical representations were generated using GraphPad Prism software (Version 7.0.0). Significance levels were set at p < 0.05 with non-significant differences denoted as "ns."

3. Results: 3.1. MTT Assay: The MTT assay was conducted to evaluate the impact of Trimethylamine N-Oxide (TMAO) and SB203580 on the viability of HepG2 cells. No significant alterations in cell viability were observed for both TMAO and SB203580 concentrations compared to the control group (Figure 1). These findings indicate that neither TMAO nor SB203580 exerted a substantial impact on the overall viability of the cells under the experimental conditions. 3.2. gRNAs insertion and vector transfection: To verify the inserted gRNAs, Colony PCR was used, and PCR products were sequenced (Figure 2). Subsequently, the plasmids containing sgRNA, along with the pcDNA3 plasmid containing Cas-13 endonuclease, were transfected into HepG2 cells using Lipofectamine LTX (Life Technologies) transfection. Successful transfection was confirmed by observing the expression of GFP protein from the pcDNA3 plasmid using a fluorescent microscope (Figure 3). 3.3. Gene Expression analysis for knockdown models: A positive control, ANXA4 (Annexin A4), was used to verify the success of the transfection processes, ensuring specific and efficient delivery of plasmids into cells. A non-targeting control was used to assess transfection-related effects in the absence of specific experimental treatments. As shown in Figure 4, HULC mRNA expression was reduced significantly compared to control group, while its expression remained unchanged in ANXA4 knockdown (KD) and non-targeting group in comparison to control group. Additionally, ANXA4 gene expression was downregulated in ANXA4(KD) compared to control group, showing ANXA4 was specifically targeted, whereas its expression was not change significantly in HULC(KD) and nontargeting groups compared to control group, indicating further validation of successful knockdown models (Figure 4). 3.4. Gene expression analysis for HULC across studied groups: As shown in Figure 5(A), HULC expression increased upon exposure to TMAO (75µM and 300µM) and this was also observed in fatty acid HepG2 cells. However, for 75µM concentration HULC expression was marginally significant in the fatty acid HepG2 cells (Figure 5B). Additionally, in comparison cells treated with 75µM, there was significant in HULC expression in cells treated 300µM concentration (P value <0.05). Conversely, neither in the HepG2 cells nor in the fatty acid HepG2 cells that HULC was knock downed TMAO did not increase the expression of HULC, indicating further confirmation of successful HULC(KD) model (Figure 5A & 5B). Moreover, there HULC was significantly downregulated in HULC(KD) cells compared to cells treated with both concentrations of TMAO (P value <0.05). 3.5. P38MAPK is inhibited by HULC knockdown P38MAPK expression was upregulated in TMAO-treated cells. Notably, there was no significant upregulation of P38MAPK expression when cells were treated with TMAO in the presence of SB203580(P38MAPK inhibitor). This effect was also observed for the HULC(KD) group. Furthermore, there was no significant changes in P38MAPK mRNA level in cells treated with SB203580 and TMAO comparted to HULC(KD) cells treated with TMAO (P value >0.05). These results suggest that HULC knockdown have the same potentiality of SB203580 to reduce mRNA level P38MAPK (Figure 6A). Moreover, there was a significant downregulation of P38MAPK in HULC(KD) and SB203580 treated cells compared to treated cells only with TMAO (P value <0.05). TNF-α and IL-6, known contributors to liver inflammation and fibrosis, were upregulated in the TMAO treatment cells in comparison to control group, and this trend persisted in the HULC(KD) group and also in the presence of SB203580 (Figure 6B; Figure 6C). Moreover, their expressions were significant in cells threated with and 300µM compared to 75µM. However, there was no meaningful changes in TNF-α and IL-6 expression in HULC(KD) cells compared to cells treated with SB203580. PNPLA3, which is associated with liver fibrosis, was upregulated in response to TMAO concentrations compared to control group, while this was not observed in the HULC(KD) group or with SB203580 treatment (Figure 6D), suggesting it is exclusively targeted by HULC/P38MAPK axis. Interestingly, PNPLA3 expression was upregulated in a dose-dependent manner upon TMAO treatment (P value <0.05). 3.6. TMAO/HULC/P38MAPK axis induced Apoptosis in HepG2 cells Apoptosis assay results explicitly showed a shift to increase apoptosis in cells treated with TMAO concentrations (P value <0.05). First, in control group there was no sign of cell death (Figure 7A). Before treating the cells with TMAO, they were threated with Oleate/palimtate combination, showing no apoptosis in this group (Figure 7B). Following this, fatty acid HepG2 cells were treated with TMAO concentrations which showed apoptosis significantly both in 75µM(Figure 7C) concentration and for 300µM (Figure 7D) in comparison to control group and Fatty acid HepG2 cells (Figure 7B). Interestingly, these changes were not found in HULC(KD)fatty acid HepG2 cells when they were treated with TMAO concentrations, 75µM (Figure 7E) and 300µM(Figure 7F).

In summary, our study not only elucidates the role of the TMAO/HULC/P38MAPK axis in NAFLD but also aligns with broader research on sarcopenia and MAPK signaling. This integrated perspective highlights the potential for therapeutic interventions targeting gut microbiota and MAPK pathways to address the interconnected pathologies of sarcopenia and NAFLD.