Introduction
Nonalcoholic fatty liver disease (NAFLD) is considered the most common type of chronic hepatic disease, and it occurs in parallel with the global obesity pandemic. During the development and progression of NAFLD, simple hepatic steatosis is considered a benign status, and steatohepatitis (NASH) is the chronic evolution pattern of hepatic steatosis associated with inflammation and fibrosis that can progressively develop into cirrhosis [
1]. The multiple-hit pathogenesis of NAFLD has been suggested to be consistently correlated with metabolic derangements in obesity in response to excessive free fatty acid (FFA) buildup and the overproduction of several cellular inflammatory mediators [
2].
Recent reports have shown that the interaction of intestinal microbiota, microbial metabolites and gut dysfunction with NAFLD is associated with the inflammatory response [
3]. Gut microbiota alterations, gut barrier dysfunction and increased gut permeability promote the translocation of bacterial materials into the liver, which might activate hepatic TLR4 activation. Moreover, TLR4 activation might cause epithelial barrier integrity loss and impair intestinal barrier function via the regulation of intestinal tight junction integrity [
4,
5]. During conditions of impaired intestinal barrier function, TLR4 activation mediates gastrointestinal-associated liver disorders. Fatty liver and increased gut permeability may generate a self-perpetuating vicious cycle in which TLR4 activation is involved, namely, gut-liver complex interrelated events in the pathogenesis of NAFLD. Hence, searching for alternative therapeutic approaches to modulate the gut-liver axis via the TLR4 signaling pathway is urgent for implications beyond NAFLD [
6].
Recently, research studies pointed out that the natural medicine product
Panax notoginseng saponins (PNS) exerted potent hepatoprotection against acute ethanol-induced liver injury and CCL
4-induced hepatic fibrosis [
7]. Previously, we proved that PNS has modulating effects on the community structure of gut microbiota [
8]. PNS could revert microbiota imbalance and reverse the higher ratio of
Firmicutes/Bacteroidetes in both diet induced obesity (DIO) mice and ob/ob mice. PNS treatment could increase the abundance of
Parabacteroides distasonis in ob/ob and DIO mice [
8], and
Parabacteroides distasonis has been proven to be negatively correlated with NAFLD and obesity [
9]. However, the underlying mechanism of PNS in obesity-induced NAFLD progression is poorly understood with respect to malfunction of the gut-liver axis. Meanwhile, the effects of PNS on the progression of NAFLD involve the TLR4 signaling pathway, and leaky gut changes need to be confirmed. Therefore, we investigated PNS regulation of hepatic steatosis and fibrosis in
lepob mice (ob/ob) and high-fat diet-induced (HFD) obese mice. Specifically, we hypothesized that PNS-modulated gut permeability inhibited the development of hepatic steatosis, inflammation and fibrosis. Here, we provide direct evidence that PNS alleviates NAFLD by decreasing hepatic lipid accumulation and inflammation in response to TLR4 activity and propose a gut-liver axis that mediates the pathogenesis of NAFLD.
Methods
Animal experiment
The experimental design and protocols were authorized by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong (CULATR NO. 4357-17). PNS was purchased from China Commercial Company and primarily contain ginsenosides Rb1, Rg1, Re, Rf, Rd and notoginsenoside R1, as shown in Fig. S1. Obese ob/ob mice from The Jackson Laboratory of Bar Harbor received a regular normal diet. Male C57BL/6J mice at 6 weeks old were fed a high-fat diet (60% fat, Research Diets, D12492) throughout the 12-week experimental period. The ob/ob and HFD mice were fed their respective diets for 4 weeks to induce obesity related NAFLD before oral administration of PNS (800 mg/kg per day) or dd water for an additional 8 weeks. The HFD and ob/ob mice were euthanized for the collection of serum, liver and small intestine tissues.
Cell treatment and transfection
An in-vitro hepatic steatosis model associated with intracellular lipid accumulation and inflammation profiles was established in cultured AML12 hepatocytes induced by palmitic acid. Then, palmitate-induced AML12 hepatocytes were treated with the vehicle or PNS (50 μg/mL) for 24 h. The real-time oxygen consumption rate (OCR) was monitored by a Agilent Seahorse XFe 24 Analyzer, and maximal respiration, proton leakage and basal respiration were calculated to assess PNS-induced dynamic metabolism. AML 12 cells were transfected with mouse siRNA targeting AMPKα 1/2 (sc-45313) or control siRNA (sc-37007) by using Lipofectamine 300 transfection reagent (Invitrogen). As a TLR4 activator, LPS (100 ng/mL) was added to AML 12 cells to detect PNS-induced AMPKα alterations.
Histology and immunofluorescence
The fixed tissues were dehydrated using a series of ethanol solutions. After cutting into 5-μm sections, the paraffin-embedded slide sections were deparaffinized and rehydrated for hematoxylin and eosin staining (H&E) and Picric Sirius red staining. For immunofluorescence staining, the rehydrated slide sections were blocked with a solution of 5% goat serum and then incubated with α-smooth muscle actin (SMA) antibody (Thermo Fisher, 14-9760-82) overnight at 4 °C in a humidified chamber. After washing, slide sections were incubated with Alexa Fluor-561-conjugated secondary antibody (Invitrogen, USA), and nuclei were stained with 4′-diamidine-2′-phenylindole hydrochloride (DAPI). Immunofluorescence results were visualized and captured via a Carl Zeiss LSM 780 system. For immunohistochemistry, slide sections were blocked with a solution of 5% goat serum and incubated with TLR4 antibody (R&D, MAB1478), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody before visualization by DAB staining. For oil red O staining, the fixed livers were dehydrated in 30% sucrose solution, embedded in OCT and sectioned at 7 μm.
Identification and quantification of SCFAs by GC–MS/MS analysis
The samples in methanol with 100 mM C2:0-d4 and 50 mM 3-methylvaleric acid internal standard were extracted by bead beating using Precellys homogenization (Bertin Technologies), followed by centrifugation. The supernatant was added with derivatization reagent (DMT-MM and octylamine in methanol) before injection. The identification and quantification of SCFAs were determined through a GC–MS/MS system equipped with SCAN and MRM mode in an Agilent 7890B GC-Agilent 7010 Triple Quadrupole Mass Spectrometer system. The separation of samples was performed through an Agilent DB-5MS capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness) with a flow rate of 1 mL/min. Experimental data were collected by using Process Cleaner and CS launcher software, and the SCFA concentrations were calculated from the standard curves established by different concentrations of chemical standards.
Quantitative real-time PCR
Total RNA was isolated from the liver tissue and purified by RNA isolated with a total RNA extraction reagent (Vazyme, R401-01), and the RNA quality was assessed by the 260 nm/280 nm measurement ratio. The synthesized cDNA was subjected to PCR, which was performed in triplicate for each sample with ChamQ SYBR color qPCR master mix (Vazyme, Q411-03) on the LC480 platform (Roche, USA). The expression of the targeted gene was normalized to β-actin. Details of the primer sequences are listed in Table S1.
Western blotting analysis
We extracted proteins from AML12 cells and liver tissues with RIPA lysis buffer, and the protein samples were subjected to centrifugation at 14,000 rpm at 4 °C for 15 min. After quantification, the protein lysates (20 μg) were separated on a 10% SDS–polyacrylamide gel. The separated protein was transferred to a membrane, and the protein membrane was blocked with 5% BSA in TBS-T solution for 2 h. After washing, the blocked membranes were immunoblotted with the targeted antibodies overnight at 4 °C and then incubated with HRP-conjugated secondary antibodies (1:1000). The reactive band signal was visually detected by ECL select substrate (GE Healthcare, Germany) on the Chemidoc chemiluminescent platform (Bio-Rad, USA).
Statistical analysis
The experimental results are presented as the mean ± standard deviation. Statistical analysis among different groups was carried out by one-way ANOVA or Student’s t-test in GraphPad Prism 8.0. Differences among different groups are shown as p-values (***p < 0.001, **p < 0.01, *p < 0.05, n.s. not significant).
Discussion
NAFLD is a benign disease associated with obesity, hyperglycemia, dyslipidemia, and insulin resistance. PNS has been described to possess anti-insulin resistance properties, ameliorate glucose intolerance and reduce obesity-induced oxidative stress [
12] in obese mice. At present, the clinical trials recorded in the U.S. National Library of Medicine have mentioned the clinical application of
Panax notoginseng in treating hyperlipidemia (NCT04069715) and obesity (NCT03654391), providing potential human evidence that supported our findings regarding use of PNS in obesity-related NAFLD. In the current study, PNS showed beneficial effects on NAFLD, as evidenced by the diminution of hepatic triglyceride accumulation and hepatic fibrogenesis in DIO and ob/ob mice. In this regard, our findings indicated that PNS influenced the main processes of fatty acid production and oxidation, which determined the flux of hepatic lipids. PNS could modulate the production of fatty acids via de novo fatty acid synthesis genes (ACC, FAS and SREBP-1C) and break them down via fatty acid oxidation genes (PPARα, CPT-1 and ACOX-1). The high level of lipid oxidation induced by PNS was also supported by the enhanced oxygen consumption rate (OCR) in PNS-treated hepatocytes under fatty acid-abundant conditions (Fig.
9).
Our mechanistic studies in HFD-induced NAFLD mice potentially implied that PNS supplementation improved hepatic steatosis and fibrosis via inhibition of hepatic CD14 and TLR4 activation. Under pathological conditions, TLR4 can induce proinflammatory signaling such as the p38 pathway [
13,
14], which induces the production of inflammatory cytokines (e.g., IL-6 and TNF-α) from Kupffer cells, ultimately leading to profibrogenic signals. Our studies showed the inhibitory effects of PNS on TLR4-mediated inflammation, which improved hepatic fibrosis in DIO mice. Furthermore, the effects of PNS on hepatic CD14 and TLR4 expression in ob/ob mice were also investigated. Despite exhibiting improvement of steatosis, PNS produced no significant change in hepatic CD14 and TLR4 expression in ob/ob mice. Furthermore, compared with the antifibrotic effects of PNS on DIO mice, PNS had less effective action on hepatic fibrosis in ob/ob mice, which might be due to the negative influence of PNS on CD14 and TLR4 activation. Previous studies have revealed that LPS-CD14-induced TLR4 activation in liver inflammation and fibrosis occurs in a leptin-dependent manner. Leptin deficiency can induce hepatic CD14 reduction in ob/ob mice, resulting in a deterioration of the Kupffer cell blockade. These findings might explain why PNS repressed fibrogenesis via inhibition of TLR4 signaling in HFD-fed mice but not ob/ob mice. Moreover, the CD14/TLR4-dependent pathway induced by PNS was not observable in ob/ob mice, demonstrating that PNS interference with TLR4/CD14 activation may be associated with leptin regulation.
Additionally, the mucosal TLR4-induced MYD88 signaling pathway contributes to the development of hepatic steatosis. To manage effective treatment options for gastrointestinal-associated liver diseases by developing new drugs, we elucidated the cross-talk between TLR4-induced inflammation and potential therapeutic medicines that facilitate the interaction between the gut and the liver. Our study verified the metabolic benefits of PNS and provided clues about the PNS-induced cross-talk between the gut and liver for protection against NAFLD.
However, whether PNS interference is due to a direct effect or is secondary to gut leakiness in decreasing hepatic steatosis and fibrosis warrants further investigation. We confirmed that PNS supplementation reduced lipid lipogenesis in palmitate-induced hepatocytes and that LPS could block PNS interference on lipid accumulation in hepatocytes. To address how the inhibition of TLR4 contributed to the reduced lipid deposition, AMPKα, as an essential cellular energy sensor in lipid metabolism, was detected. Our results showed that PNS-induced AMPKα activation reduced lipogenesis in hepatocytes and that this promotion could be interrupted by TLR4 activation, which means that the inhibitory effects of PNS on TLR4 have beneficial effects on AMPKα regulation, resulting in the improvement of hepatic lipid metabolism. Next, our research aimed to confirm whether PNS modulation of the TLR4 signaling pathway involved gut-liver axis malfunction. Accumulating evidence in both animals and humans has indicated that increased intestinal permeability (leaky gut) facilitates the translocation of microbial products, including SCFAs, across tight junctions into the liver [
15], which could trigger the progression of NAFLD [
16,
17]. Our studies confirmed that the total contents of FFAs and TG were decreased after PNS treatment, which suggests that PNS can induce FFA changes to improve NAFLD. The significant differences in the gut levels of LCFAs (oleic acid, 2-palmitoyglycerol) and SCFAs (acetic acid, butyric acid, propionic acid, etc.) after PNS exposure in HFD-fed mice suggested that PNS might influence the changes in the fatty acid composition and content in the gut of mice. A notable finding of this study was the PNS-induced decreasing trend in hepatic SCFAs, as well as increased SCFA production in the small intestine observed in DIO-induced NAFLD mice, which indicated that an indirect effect of PNS on NAFLD progression was associated with gut permeability improvement by increasing the expression of tight junction-associated proteins (ZO-1 and Claudin-1). The reason that PNS increased the levels of SCFAs in the gut might be associated with the regulation of PNS on increasing the level of SCFA-producing bacteria. Due to the improvement of leaky gut induced by PNS, the large amount of SCFAs has difficulty entering the liver to activate G-protein coupling receptors such as GPR41, further mediating steatosis and inflammation [
18]. In the current studies, our results from HFD mice underscored a contributing role of TLR4 activation in elevating gut leakiness [
4,
19], given that PNS decreased TLR4 expression and activated tight junction proteins (ZO-1 and claudin-1). We observed a significant up-regulation of TLR4 expression in the intestine of LPS-treated mice in the presence of PNS, suggesting that LPS can reverse the inhibitory effect of PNS on TLR4 expression in the intestine. Indeed, LPS treatment can activate TLR4, which was responsible for the increase of gut permeability [
20]
. LPS treatment abolished the improvement of gut permeability by PNS. Although the recovery of TLR4 expression in PNS-treated mice using LPS as an antagonist of PNS can completely conclude that the effect of PNS is solely dependent on TLR4 activation, as LPS may have some off-target effect, the findings of our study combined with literature report may somehow indicate that the improvement of gut permeability induced by PNS is, partially if not all, associated with PNS mediated-TLR4 deactivation. Indeed, PNS modulation was relevant in the activation of TLR4 elicited by LPS exposure in both liver and intestinal tissues of HFD mice, as supported by the results that LPS weakened the modulatory effects of PNS on NAFLD. LPS exposure in HFD mice with PNS administration led to exacerbated steatosis by decreasing AMPKα activation and increasing collagen I, collagen IV and α-SMA, which reversed the antifibrotic activation of PNS. The interference of LPS on the anti-NAFLD effect of PNS confirmed that the improvement of NAFLD induced by PNS was associated with the TLR4 pathway.
Acknowledgements
Metabolomics technical assistance was supported by the University of Hong Kong Li Ka Shing Faculty of Medicine Centre for PanorOmic Sciences Proteomics and Metabolomics Core. The study was financially supported by grants from the research council of the University of Hong Kong (Project Codes: 104004092 and 104003919), Gala Family Trust (Project Code: 200007008), Government-Matching Grant Scheme (Project Code: 207060411), Contract Research (Project cord: 260007830, 260007482), Health and Medical Research Fund (Project code: 15162961) and Health and Medical Research Fund (Project code: 16172751).
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