Introduction
Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the world, and the incidence is increasing year by year [
1]. NASH is based on inflammation, liver steatosis, liver cell damage and fibrosis of varying degrees, which can progress to liver cirrhosis or even liver cancer [
2]. However, there is a lack of clinically effective radical drugs at the moment. Therefore, there is an urgent need to have a deeper understanding of the etiopathogenesis of NASH, and to find effective treatment drugs to alleviate the pathogenesis.
Autophagy, a self-degradation process of cells, which degrade cellular proteins and damaged or excessive organelles through the formation of double membrane autophagosomes, plays a role in energy balance and cytoplasmic quality control [
3,
4]. In recent years, increasing evidence observed that the autophagy flux is blocked in the livers of NASH patients [
5,
6]. Additionally, autophagy defects in hepatic sinusoidal endothelial cells in patients with NASH promote the development of early liver inflammation, endothelial-mesenchymal transition, apoptosis and liver fibrosis in NASH [
7]. Thioredoxin-interactingprotein, a key mediator of cellular stress response, directly interacted with p-PRKAA, leading to inactivation of mTOR and nuclear translocation of TFEB, thereby promoting autophagy and improving liver steatosis, inflammation and fibrosis [
8]. All these evidences demonstrated that autophagy is closely related to liver disease, which is a potential new therapeutic target. However, the mechanism of how autophagy regulates NASH patients remains unclear.
Pyroptosis is a new type of inflammatory cell death, which mainly relies on the inflammasome composed of NLR family pyrin domain containing 3 (NLRP3), ASC and pro-caspase 1 [
9]. In NASH, lipotoxicity, organelle stress, and liver cell death triggered inflammasome activation, while excessive activation of NLRP3 inflammasomes exacerbated liver steatosis [
10]. Moreover, the activation of NLRP3 inflammasome leaded to pyroptosis of mouse and human primary hepatocytes, promoted IL-1β secretion, and induced stellate cell activation and liver fibrosis [
11]. In recent years, the regulatory relationship between autophagy and pyroptosis has been reported. For example, inhibition of autophagy could induce LDH release, NLRP3 inflammasome activation and pyroptosis [
12]. Liraglutide ameliorates NASH by prohibiting NLRP3 inflammasome and pyroptosis through mitophagy activation [
13]. The above studies indicated that the process of autophagy inhibiting pyroptosis plays a crucial part in the progress of NASH. However, the molecular mechanism regulating the “autophagy / pyroptosis signal pathway” is still poorly understood. Therefore, studying this signal pathway will help us to understand the pathogenesis of NASH, which is of great significance for finding new treatment strategies for NASH.
tRNA-derived small RNA (tsRNA) is a novel regulatory non coding small RNA, which exert an important influence in multifarious biological processes [
14]. tsRNA has been reported to be involved in the process of many diseases, including various cancers, acute cerebral infarction, bone metabolism related diseases, etc. [
15]. Currently, a study has reported that tsRNA plays an important role in non-alcoholic fatty liver disease (NAFLD) [
16]. Simultaneously, we also proposed that tsRNA plays a vital role in NAFLD cell models and mouse models via regulating autophagy [
17]. However, the specific role and molecular mechanism of tsRNA in NASH remain to be elucidated.
In recent years, as an alternative to drug intervention, dietary strategies to improve NASH have been increasingly popular. In previous research, we manifested that blueberry significantly reduced liver cell apoptosis and fat accumulation, enhanced lipid metabolism and improved fatty liver, nevertheless, it is not yet clear which active ingredients the blueberry mainly uses to improve fatty liver [
18‐
20]. It has been reported that cyanidin-3-O-glucoside (C3G), one of the most abundant monomers in vaccinium oxycoccus pigment, improved hepatic steatosis in mice [
21,
22]. Furthermore, tectorigenin (TEC) was also one of the active ingredients of blueberries, which inhibited the expression of toll-like receptor-4 (TLR4) and the activation of the MAPK/NF-κB pathway to promote autophagy and protect liver failure [
23]. These analysis emphasized the need to estimate the reasonable utility of blueberry monomer in NASH treatment and to research the mechanisms involved. Herein, we further explore which active substances in blueberries can improve the function of NASH by autophagy and pyroptosis; and focus on whether blueberry monomer relies on tsRNA to regulate the connection between autophagy and pyroptosis, in order to provide safe and effective potential drugs for NASH treatment, and provide a strong theoretical and experimental basis for the prevention and treatment of NASH.
Materials and methods
First, we smashed the blueberries with a homogenizer. Then, we took 0.5 g blueberry homogenate and used 5 ml methanol solution (Fisher) containing 0.1% formic acid (Sigma-Aldrich) for ultrasonic extraction for 15 min. Next, centrifuged for 20 min at 10 °C and 12,000 r/min, and take the supernatant. Finally, the liquid phase was detected after filtration by microporous membrane (0.45 μm).
Detection of monomers by ultra performance liquid chromatography combined-mass spectrometry (UPLC-MS)
We extracted monomers from blueberries with methanol and distilled water respectively. When extracting with methanol, blueberry and 0.1% formic acid methanol were 1:4, then grind in a mortar. Next, the homogenate was centrifuged at 3500 rpm/min for 5 min, and took the supernatant into the liquid phase for detection, and the injection volume was 1 μl. Water extraction: blueberry and distilled water was 1:4, and the remaining steps were consistent. Finally, blueberry monomers were prepared into 1000 ppb, 800, 600, 400, 200 and 100 ppb with 0.1% formic acid methanol solution. The standard curve was drawn according to the peak area and concentration by UPLC-MS.
Cell culture and treatment
Human hepatoma cell line HepG2 was obtained from the Chinese Academy of Sciences (CAS). HepG2 cells were cultured in DMEM (Dulbecco’s modifified Eagle’s medium) added with 10% fetal bovine serum (Biological Industries, 1707254) and antibiotics (Hyclone, HYC-SV30010) under a cell incubator with 5% CO2 at 37 °C.
In order to model NASH cells, HepG2 cells were cultured with 0.5 mM concentration of FFA [
24]. In experimental group, NASH cell model were treated with 25, 50 and 75 μM TEC, respectively, and the selection of TEC concentration was based on others report [
25]. To block autophagy, hepatocytes were cultured with 5 mM 3-Methyladenine (3MA) [
26]. To knockdown tRF-47-58ZZJQJYSWRYVMMV5BO (tRF-47) in vitro, tRF-47 antagomir was transfected into TEC-treated steatosis hepatocytes using Lipofectamine 2000 (Invitrogen, USA).
Cell viability assay
The Cell Counting Kit-8 (CCK8, Dojindo, CK04) was utilized to assess the viability of HepG2 cells. Cells were cultured with FFA in 96-well plates (1 × 104 cells/well) and treated with different doses of TEC for 24 h. Then, we added CCK8 reagent, and put the plate into 37 °C incubator for 4 h. The absorbances in different groups were detected at 450 nm. In the blank group, the well only contained medium, and the cells without any treatment were used as the control group.
Western blot analysis
We lysed cells with RIPA buffer (beyotime, China) and extracted proteins from cells. Then, BCA assay kit (Invitrogen) measured the protein concentration. 10% SDS-PAGE was employed to separate proteins. The protein was transferred to the PVDF membrane (Bio-Rad, USA), the PVDF membrane was blocked with 5% milk, and then incubated with a primary antibody. And then incubated with an NLRP3 (Abcam; ab263899; dilution 1:1000); GSDME (Abcam; ab215191; dilution 1:1000); or GAPDH antibody (Abcam; ab181602; dilution 1:10,000) at 4 °C overnight. After washing the membrane, it was incubated with goat anti-rabbit IgG-HRP for 1–2 h. The photos were taken using Odyssey infrared Fluorescence Western blotting system (Li-Cor Biosciences), and the quantitative pictures was obtained via ImageJ software.
LDH release assay
According to the manufacturer’s protocol, LDH levels in the cell supernatants were determined using an LDH Cytotoxicity Assay Kit (Beyotime, C0016). The absorbance was read at 490 nm with a microplate reader (Thermo Fisher Scientific).
RNA isolation, small RNA libraries construction, and sequencing
The total RNA was obtained from the FFA group and the FFA + TEC group of HepG2 cells (n = 3 for each group) by the RNeasy Mini Kit (Qiagen, Hilden, Germany). The quality of RNA was detected by the 1% agarose gel electrophoresis. The concentration and integrity of total RNA were measured by the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Following passing the quality control tests, the Multiplex Small RNA Library Prep Set for Illumina (NEB, MA, USA) was leveraged to establish the library following the manufacturer’s protocol. Briefly, the rRNA was removed and the remaining RNA was cut into small pieces. RNase H-reverse transcriptase (NEB, MA, USA) was used to synthesize the first-strand cDNA and the end repair was conducted, then the fragments were sorted and PCR was performed to amplify the cDNA. An Illumina Hiseq2500 platform (Illumina, San Diego, California) was used for the sequencing of library preparations.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA were extracted using Trizol reagent (Invitrogen, CA, USA) and subjected to obtain cDNA by PrimeScript™ RT reagent kit (Takara, China). qRT-PCR analysis was performed using Fast SYBR Green master mix (Life Technologies, USA). U6 and GAPDH served as an internal control. The qRT-PCR conditions used were: 1 cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 5 s, and annealing and extension at 57 °C for 30 s. The relative expression were calculated using the 2
−∆∆Ct method. The primer sequences were presented in Additional file
1: Table S1.
Animals and treatment
The male C57BL/6 mice (8–10 week old, 20–25 g) were purchased from the Animal Center of Guiyang Medical College, and randomly divided into 5 groups (n = 5). The animal model was constructed based on the method recently described by others [
23,
27]. The first group was the control group: mice were fed with standard feed. The second group was the NASH group: mice were fed with the compound high fat feed (88.3% common feed + 10% lard + 1.5% cholesterol + 0.2% sodium cholate). The remaining three groups were low, medium and high drug groups: the mice were administered with TEC by gavage at 7.5, 15.0 or 30.0 mg/kg once per day for 6 weeks after 10 weeks of high fat feeding [
27,
28]. To knockdown tRF-47 in vivo, tRF-47 antagomir (5 μg/mouse in 1.5 mL saline) was injected into the tail vein of NASH mice 3 times per week for 2 weeks after 9 weeks of high fat feeding [
17]. At the end of the experimental period, the animals were euthanized, and the livers and serum were immediately processed for pathological examinations and biochemical analyses.
Biochemical analysis
The serum samples were used for biochemical analysis. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by a Biochemical Analyzer (Siemens Advia 1650, Germany). The level of malondialdehyde (MDA) was detected by the MDA kit (Nanjing Jiancheng, China).
Haematoxylin–eosin (H&E) staining
Liver tissues were fixed in 10% neutral buffered formalin, dehydrated in graded volumes of ethanol alcohols, embedded in paraffin, sectioned at 3 μm slices, and stained with hematoxylin and eosin (H&E) for microscopic examination.
Triglyceride content detection
Triglyceride (TG) detection kit (Nanjing Jiancheng, China) was used to analyze the level of liver TG. Briefly, a small portion of liver tissue (50 mg) was collected and homogenized in 100% ethanol (450 ml). After centrifugation, glycerol lipase oxidase method was used for analysis. Samples were reacted with the mixture from the kit and were incubated at 37 °C for 10 min, and absorbance at 510 nm was read with a microplate reader (Thermo Fisher Scientific).
Total cholesterol content detection
Total cholesterol (TC) levels in liver were analyzed using a CheKine™ Total Cholesterol Colorimetric Assay Kit (Abbkine, KTB2220). Liver tissues were harvested and lysed with the buffer. Subsequent procedure was performed with the kit according to protocol and the samples were read with spectrometer at 500 nm. Before measurement, we first standardize the cholesterol concentration and draw the standard curve.
Immunohistochemistry
Immunohistochemistry for NLRP3 and gasdermin E (GSDME) was conducted, the tissues sections were blocked with 8% goat serum in PBS, and then incubated with anti-NLRP3 antibody (Abcam, ab214185, dilution 1:100) and anti-GSDME antibody (Abcam, ab230482, dilution 1:500) at 4 ˚C for 12 h. Thereafter, the sections were incubated with anti-rabbit IgG H&L (HRP) (Abcam, ab6721, dilution 1:1000) at room temperature for 1 h. Finally, the sections were examined under a light microscope and Nikon Photo-Imaging system (H550L, Tokyo, Japan).
Oil Red O staining analysis
The Oil Red O Kit was utilized to stain TG. 5 μm thick frozen sections of liver tissue or HepG2 cells were washed twice with PBS and fixed in 4% paraformaldehyde at room temperature for 40 min. After two washes in 60% isopropyl alcohol, the slices or cells were stained with an Oil Red O stain kit (KeyGEN, KGA329) according to the manufacturer’s protocol. After removing the Oil Red O solution, the slices or cells were washed with distilled water five times and observed under a microscope.
Enzyme-linked immunosorbent assays
Following the instruction of the ELISA kit (Mlbio), we used the enzyme-linked immunosorbent assays to quantify IL-6, TNF-α, IL-10, IL-4, IL-17, and IL-1β levels in liver tissue or HepG2 cell supernatants.
Analysis of immunofluorescently-stained
The liver tissue or HepG2 cells were fixed with 4% paraformaldehyde. The samples were blocked with 5% BSA in PBS at room temperature (RT) for 1 h, and then incubated with rabbit monoclonal anti-LC3B IgG (Abcam, EPR18709) at 1 µg/ml overnight. This was followed by incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Abcam, ab150077) at RT for 1 h. 4’, 6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. We used a microscope (Olympus Corporation) to view the samples.
Statistical analysis
The data were expressed as the mean ± standard deviation (SD). All the groups were compared using analysis of one-way analysis of variance (ANOVA) or t-test. SPSS 17.0 (SPSS, USA) statistical software and Graphpad Prism 8.0. (La Jolla, USA) were used to analyze these data, and p < 0.05 was regarded as statistically significant.
Discussion
NASH is an emerging risk factor for type 2 diabetes and end-stage renal disease [
31]. It has become the second leading cause of liver disease among adults waiting for liver transplantation in the United States, which is expected to become the most common cause of liver transplantation in the next ten years [
32]. However, there are still many doubts about the cause of NASH, and there is a lack of clinically effective drugs. Therefore, it is urgent to study the pathogenesis of NASH from different angles and find new drugs to lay the foundation for the clinical treatment of NASH. In this study, TEC, one of blueberry monomers, effectively ameliorated the symptoms of NASH by enhancing the expression of tRF-47, including reducing lipid accumulation, activating autophagy, and inhibiting pyroptosis. Our research clarified the pathogenesis of NASH from a new perspective and provided a reliable scientific basis for targeted therapy of NASH.
Currently, nano based therapy has been widely used to treat a variety of diseases [
33], and dietary recommendations and physical exercise remain the mainstay of NASH therapy. Happily, compounds extracted from natural products are increasingly being used in NASH because of their efficacy and low side effects [
34]. Many evidence proved that vaccinium oxycoccus pigment could be used in the treatment of obesity and NASH, such as G3G, whereas other monomers of vaccinium oxycoccus pigment also have similar effects [
35]. TEC has reported to inhibit adipogenesis and related genes transcription [
25]. Unfortunately, the regulatory effect of TEC on NASH has not been reported. In our study, both C3G and TEC could significantly inhibit the formation of lipid droplets in steatotic HepG2 cells, but the effect of TEC on the formation of lipid droplets was significantly higher than that of C3G, which proved that TEC is a potential therapeutic agent for the treatment of NASH.
NASH is mainly characterized by excessive fat deposition in the liver and dysregulation of lipid metabolism and reactive oxygen [
36,
37]. Many studies have proved that autophagy and hepatic lipid metabolism were interrelated [
5]. Singh et al. [
38] reported that autophagy mediates lipid metabolism by reducing the production of triglycerides and effectively prevents the development of hepatic steatosis. In addition, autophagy deficiency could contribute to oxidative stress, cause mitochondrial dysfunction, and accelerate the activation of NLRP3 inflammatory, which resulting in pyroptosis [
39,
40]. TEC effectively inhibited palmitate-induced reactive oxygen species production and mitochondrial membrane potential collapse [
41]. In this study, lipid accumulation and increased abundance of LC3B by activated pyroptosis (over-expressed NLRP3 and GSDME) were observed in vitro NASH model, which were consistent with the previously reported work related to impaired hepatic autophagy is associated with NASH [
6]. Furthermore, in addition to alleviating the symptoms of lipid deposition in NASH models, TEC also were observed activating autophagy (increased LC3B expression), inhibiting pyroptosis and reducing the expression of inflammatory factor (IL-6, TNF-α, IL-10, IL-4, IL-17, and IL-1β) and TLR4, which is similar to other studies [
23,
42,
43]. Considering the relationship between autophagy and pyroptosis, we speculated that TEC may treat NASH via regulating autophagy and pyroptosis. Consistently, autophagy inhibitors could counteract the inhibitory effect of TEC on lipid droplet formation and enhance the expression of pyroptosis protein and release of LDH. Combined with TEC improving liver failure by regulating autophagy pathway, and TEC regulated TLR4, NF-κB and MAPK pathway to inhibit the occurrence of inflammatory response, which all proved our view that TEC may improve NASH by regulating hepatocyte autophagy and pyroptosis [
23,
44].
tsRNA is a non-coding small RNA which involved in a variety of physiological and pathological processes. It has been identified as a new class of NAFLD biomarkers due to the significant increase of tsRNAs in plasma of NAFLD patients [
45]. Interestingly, we have previously reported that tsRNAs regulated autophagy, which played an important role in NASH [
17]. In the present study, we sequenced the small RNA of hepatocytes with TEC and FFA treated, and found 122 differentially expressed tsRNAs, including 78 up-regulated and 44 down-regulated tsRNAs. These differential tsRNAs were mainly involved in “PI3K-Akt signaling pathway”, “Wnt signaling pathway”, “MAPK signaling pathway” and “FoxO signaling pathway”, which have been reported to be participated in autophagy and pyroptosis [
46]. We then screened the tsRNAs involved in autophagy and pyroptosis signaling pathway for PCR verification, which showed that the expression difference of tRF-47 was the most significant after TEC treatment. Not surprisingly, tRF-47 antagomir significantly reversed the regulatory effects of TEC on autophagy and pyroptosis in vitro. Similarly, in vivo experiments have manifested that TEC improved NASH by activating tRF-47 molecule. These results demonstrated that TEC enhanced autophagy and weaken pyroptosis by activating the expression of tRF-47, ultimately reducing lipid deposition and improving NASH.
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