Tilianin Protects against Nonalcoholic Fatty Liver Disease in Early Obesity Mice

Abstract

Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the most frequent types of liver disease in pediatric populations with obesity. Tilianin has multiple biological activities including anti-inflammatory and antioxidant. Here, we aim to explore the functions and possible mechanisms of tilianin on NAFLD in obese children. A high-fat high-carbohydrate (HFHC) diet was used to feed 21-d-old mice. Tilianin was administered at a dose of 10 or 20 mg/kg daily. HFHC-fed mice gained weight, increased liver index. The liver showed hepatocyte ballooning, inflammatory infiltration, and steatosis. Elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) and reduced the high-density lipoprotein cholesterol (HDL-C) level were found in HFHC-fed mice. Administration of tilianin significantly reduced these impairments. We further evaluated proteins related to lipid metabolism and observed that LXRα, SREBP-1c, FAS and ACC1 expression were blunted following tilianin administration. In addition, tilianin suppressed reactive oxygen species (ROS) overproduction and lipid peroxide 4-Hydroxynonenal expression, ascribed to its oxidative stress-modulating capacity. Tilianin also reversed the increase in F4/80 expression and proinflammatory cytokine levels. Of note, tilianin administration resulted in decreased protein levels of active caspase-1 and NOD-like receptor protein 3 (NLRP3) in HFHC-fed mice. Our study suggests that tilianin may ameliorate NAFLD in early obese mice by modulating lipids metabolism, oxidative stress, and inflammation, which may in part involve inhibiting NLRP3 inflammasome activation.

INTRODUCTION

Childhood obesity rates have increased rapidly in recent years in countries of all economic levels. Childhood overweight and obesity is an undeniable global health crisis.¹⁾ Unhealthy dietary habits and physical inactivity are the leading causes of childhood obesity. Obese children are more likely to overweight when they grow up and have greater susceptibility to develop chronic diseases related to metabolic disorders than peers with a healthy weight.²⁾

Nonalcoholic fatty liver disease (NAFLD) is a disorder associated with metabolic dysfunction manifested as steatosis, which occurs in individuals who do not drink excessively.³⁾ In light of the massive obesity epidemic in children, NAFLD has emerged as a frequent type of liver disorder in pediatric populations.⁴⁾ Early steatosis and more severe subgroup non-alcoholic steatohepatitis (NASH) are encompassed in the pathological progress of NAFLD, some patients are at risk of developing a succession of severe liver diseases, even liver cancer.⁵⁾ The biological mechanisms of NAFLD progression are important and unresolved problems. The ‘two hit’ model is a most popular proposed hypothesis, the first is the excessive accumulation of triglycerides in liver cells (steatosis), and the second is the triggering of NASH through other factors, such as lipid peroxidation and lipotoxicity.⁶⁾ Moreover, Oxidative stress is considered to be a pathway of hepatocyte injury and is closely related to inflammation and NASH onset.⁷⁾

NOD-like receptor protein 3 (NLRP3) inflammasome is widely reported as a factor that induces inflammation in NAFLD.^8,9) The sensor molecule NLRP3, aptamer protein ASC and effector molecule pro-caspase-1 constitute NLRP3 inflammasome, a cytoplasmic multiprotein scaffold. In response to danger signals, NLRP3 aggregates with ASC and pro-caspase-1 to form the NLRP3 inflammasome, thereby pro-caspase-1 is activated and subsequently induces interleukin (IL)-18 and IL-1β maturation and release.¹⁰⁾ Substantial evidences link NAFLD development and progression to NLRP3 inflammasome- and inflammatory factor-related responses.¹¹⁾

Tilianin (acacetin 7-O-β-D-glucoside) is a steroidal alkaloid identified in various Chinese traditional herbal plants,¹²⁾ its molecular structure is shown in Fig. 1A. Substantial evidences have suggested that tilianin have hypotensive, anti-diabetic and cardioprotective properties.^13,14) Previous study showed that tilianin exerted antihyperlipidemic effect by regulating the proinflammatory profile and energy metabolism pathway.¹³⁾ Furthermore, tilianin was shown to reduce reactive oxygen species (ROS) production and modulate oxidative stress.¹⁴⁾ A recent report indicated that acacetin, an aglycone of tilianin, alleviated NAFLD by modulating lipid metabolism and inflammation in obese mice.¹⁵⁾ However, whether tilianin improves NAFLD and its mechanisms have not been elucidated. Thus, we examined how tilianin modulates NAFLD-induced aberrant lipid metabolism, oxidative stress, and inflammatory responses, and clarified their possible mechanisms of action in relation to the NLRP3 inflammasome in a young mouse model.

Fig. 1. Tilianin Inhibited Weight Gain of HFHC-Fed Mice

(A) Molecular structure of tilianin. (B) Experimental procedures for mouse model establishment and tilianin administration. The control mice fed standard diet. Mice were fed a HFHC diet for 12 weeks and administered vehicle and 10 mg/kg tilianin (Tilianin-L) or 20 mg/kg tilianin (Tilianin-H) by intragastric administration (i.g.) once daily from 5th week. (C) Body weight was monitored every week. * p < 0.05, ** p < 0.01 compared to the control group; ^# p < 0.05, ^## p < 0.01 compared to the HFHC group. (D) BMI (Body Mass Index) was calculated at the end of experiment.

MATERIALS AND METHODS

Animals

Animal experiments were approved by the Ethics Committee of the Anhui Medical University (No. LLSC20221131) and followed the standards of the Guide for the Care and Use of Laboratory Animals. Twenty-one-day-old male C57BL/6 mice were purchased from Changsheng Biotechnology (Shenyang, China) housed in cages on a 12-h day-night cycle with food and water ad libitum. Ambient temperature and humidity were controlled at 22 ± 1 °C and 45–55%, respectively. Mice were acclimated for a week before the initiation of the study.

Animal Treatment

Mice were randomly allocated to four groups (Fig. 1B), including control group, high-fat high-carbohydrate (HFHC) group, HFHC + 10 mg/kg/d tilianin (HFHC + Tilianin-L) group, HFHC + 20 mg/kg/d tilianin (HFHC + Tilianin-H) group.

The control group mice fed with a standard chow diet; mice in HFHC group were fed a HFHC diet. Based on the previously described method,¹⁶⁾ different sources of HFHC diet were used. Briefly, the HFHC diet consisted of a high-fat diet (Dyets, Wuxi, China) and 42 g/L carbohydrate, which contained 55% fructose (Xiangchi Jiangyuan Biotechnology, Binzhou, China) and 45% sucrose (Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjing, China). The tilianin-L and tilianin-H groups comprised mice fed HFHC diet and administered with 10 or 20 mg/kg tilianin by gavage.^17,18) Tilianin (purity ≥98) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (#T889422, Shanghai, China).

The mice in HFHC, HFHC + Tilianin-L, HFHC + Tilianin-H groups were fed the HFHC diet for 4 weeks and then treated with vehicle or tilianin once a day for 8 weeks. Body weight was recorded weekly and naso-anal length was measured at 12 weeks. Body mass index (BMI) is equal to body weight (g) divided by the square of the naso-anal length (cm). Liver index is liver weight as a percentage of body weight. At the end of experiments, the blood and liver samples were collected and frozen.

Assay for the Inflammatory Cytokines

The levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6 and IL-18 in serum and/or liver were measured using the commercial enzyme-linked immunosorbent assay (ELISA) kits (MultiSciences Biotech, Hangzhou, China) according to the instruction provided by the manufacturer.

Assay for the Biochemical Parameters

The serum of mice was collected for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT). Liver tissue was homogenized to determine the levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C).¹⁹⁾ The above biochemical parameters were measured using the kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Oil Red O Staining

Liver tissues were fixed and then dehydrated in 20 and 30% sucrose solution. Livers were embedded with OCT and sliced at 10 µm in a cryostat (Leica Biosystems, Nussloch, Germany). The sections were immersed in 60% isopropanol, followed by staining with Oil Red O (Merck, Billerica, MA, U.S.A.). Then, the cryosections were stained with hematoxylin (Solarbio, Beijing, China) after washing with distilled water.

Hematoxylin–Eosin (H&E) Staining

Fixed liver was embedded and prepared into 5 µm slices. The liver sections were stained with H&E and the photomicrograph was captured using Olympus DP73 system (Olympus, Tokyo, Japan).

ROS Examination

The OCT-embedded liver was sectioned into 10 µm, and the ROS was assessed using active oxygen detection kit (BBcellProbe, Shanghai, China) with the fluorescent probe dihydroethidium (DHE) on the basis of manufacturer protocols.

Immunofluorescence

Paraffin-embedded livers were prepared into 5-µm sections and then immersed in alcohol and washed with phosphate buffered saline (PBS). Antigen retrieval was performed before sections were blocked with 1% bovine serum albumin (BSA) (Sangon Biotech, Shanghai, China) for 15 min. Primary antibody against F4/80 (1 : 100; #DF2789, Affbiotech, Changzhou, China) were then added on the sections and incubated at 4 °C. The next day, the livers were incubated with goat anti-Rabbit immunoglobulin G (IgG) (1 : 200, #A27039, Invitrogen, Carlsbad, CA, U.S.A.), and finally stained with 4′-6-diamidino-2-phenylindole (DAPI) (Aladdin, Shanghai, China).

Immunohistochemical (IHC) Staining

The embedded liver paraffin blocks were made into 5 µm sections, deparaffinized, and rehydrated by different concentrations of alcohol. For antigen retrieval, the sections were boiled in the retrieval buffer. Chilled slides were immersed in 3% H₂O₂ for blocking endogenous peroxidase. Then, the slides were blocked for 15 min in 1% BSA and incubated overnight at 4 °C with 4-Hydroxynonenal (4-HNE) primary antibody (#MAB3249-SP; R&D Systems, MN, U.S.A.) diluted 1 : 100 in PBS. The secondary antibody was HRP-conjugated goat anti-mouse IgG (#31430; ThermoFisher Scientific, Pittsburgh, PA, U.S.A.). The liver slides were visualized with dimethylaminoazobenzene (DAB) and hematoxylin.

Western Blotting

RIPA buffer (Solarbio, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) was employed to extract total liver protein on ice. After quantification of protein concentration, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck, Billerica, MA, U.S.A.). After blocking and washing in PBS, the membranes were incubated with primary antibodies overnight at 4 °C, including ACC1 (1 : 500; #A19627, ABclonal Biotechnology, Wuhan, China), FAS (1 : 400; #P25445, Affbiotech, Changzhou, China), caspase-1 (1 : 1000; #WL03325, Wanleibio, Shenyanag, China), SREBP-1c (1 : 500; #AF4728, Affbiotech), NLRP3 (1 : 1000; #A5652, ABclonal Biotechnology, Wuhan, China), LXRα (1 : 1000; #A2141, ABclonal Biotechnology), as well as the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 10000; #60004-1, Proteintech, Wuhan, China). The membranes were then incubated with secondary antibodies (#SE134, #SE131; Solarbio) at 1 : 3000 dilution for 1 h. Lastly, ECL (Solarbio) was used to detect the bands.

Patients

To demonstrate the characteristics of children with NAFLD, we recruited 29 patients (17 boys and 12 girls) from Anhui Medical University. The inclusion criteria were 8–17 years of age, obesity, and NAFLD. Patients treated with medications that could affect insulin, glucose, transaminases, or cholesterol levels were excluded from the study. Simultaneously, 30 non-obese children (17 boys and 13 girls) with no body organ pathology were recruited as controls. Blood samples were collected from the subjects for the detection of IL-1β and IL-18 levels. The study was approved by the Ethics Committee of the Anhui Medical University [No. PJ-YX2022-005] in accordance with the Declaration of Helsinki and informed consent of the patients’ parents was obtained. The description of the baseline characteristics subjects was shown in Supplementary Table S1.

Statistics

All results are presented as mean ± standard deviation (S.D.). Significant differences between two groups were analyzed with Student’s t-test. ANOVA followed by Tukey’s multiple comparisons test was applied for multiple comparisons. Values of p < 0.05 was identified as statistically significant unless otherwise specified.

RESULTS

Tilianin Reduced Obesity of HFHC-Fed Mice

The values of body mass index (BMI) and waistline were significantly higher in children with NAFLD compared to those without NAFLD (Supplementary Table S1). Similarly, HFHC diet resulted in significant weight gain in mice (Fig. 1C). The high dose tilianin treatment suppressed the body weight gain in HFHC-fed mice, being more effective than the low dose tilianin treatment. The BMI was higher in HFHC-fed mice than that in control group (Fig. 1D). BMI of tilianin groups tend to be lower than that of HFHC group, though not reaching statistical significance.

Tilianin Protected against Liver Injury in HFHC-Fed Mice

Serum ALT and AST levels are key indicators for evaluation of acute liver damage. Children with NAFLD had higher serum levels of ALT, AST and GGT (Supplementary Table S1). Consistently, the levels of ALT and AST were elevated in the HFHC-fed mice (Figs. 2A, B), while tilianin administration reduced these raised levels. HFHC diet also increased the serum levels of GGT (Fig. 2C). Treating the mice with tilianin decreased GGT level caused by HFHC diet feeding. Liver specimen from normal group was showed normal color and shape, as well as smooth surface (Fig. 2D). However, the liver appearance in the HFHC-fed mice was rough and covered with particles, visually paler. Tilianin improved these pathological appearances of liver. In addition, mice fed HFHC diet exhibited a higher liver index than that in normal diet-mice (Fig. 2E), and the liver index was reduced by tilianin administration. H&E staining of liver from HFHC-fed mice exhibited large fat droplets, inflammatory cells infiltrated and vacuolar degeneration (Fig. 2F). After the administration of tilianin, the pathological damage of liver tissues had different degrees of improvement. These results indicated that tilianin alleviated liver injury in HFHC-fed mice.

Fig. 2. Tilianin Alleviated Liver Injury in HFHC-Fed Mice

The serum levels of (A) ALT, (B) AST and (C) GGT in mice. (D) Liver specimens. (E) Liver index. (F) Representative HE-stained liver sections.

Tilianin Ameliorated Abnormal Lipid Metabolism and Oxidative Stress in HFHC-Fed Mice

The TC and LDL-C levels measured in children with NAFLD was higher than that in normal children (Supplementary Table S1). Correspondingly, the mice in HFHC group had increased levels of LDL-C and TC, reduced HDL-C level (Figs. 3A–C). The administration of tilianin reversed these abnormal levels. Notably, liver histology showing fat deposition with Oil Red O staining revealed an increase in lipid accumulation in HFHC-fed mice (Fig. 3D). The lipid accumulation was reduced in livers of mice administrated tilianin. In contrast to the lipid staining, liver expressions of lipid metabolism-related proteins were increased in HFHC-fed mice (Figs. 3E–I), including ACC1, FAS, SREBP-1c and LXRα, and were reduced after tilianin treatment, especially at high doses. These results demonstrated that tilianin reduced lipid accumulation may through downregulating the expression of FAS, ACC1, SREBP-1c and LXRα.

Fig. 3. Tilianin Attenuated Aberrant Lipid Metabolism and Oxidative Stress in HFHC-Fed Mice

The levels of (A) LDL-C, (B) HDL-C, and (C) TC in the liver. (D) Representative liver sections stained with Oil Red O. (E–I) The protein expression levels of ACC1, FAS, SREBP-1c and LXRα in the liver were analyzed by Western blotting. (J) ROS concentration in mice detected using DHE probe. (K) 4-HNE expression was analyzed by IHC analysis.

Additionally, the concentration of ROS in livers was increased in HFHC-fed mice (Fig. 3J), which was reduced by tilianin treatment. IHC analysis revealed the elevated level of the oxidative damage marker 4-HNE in the HFHC group (Fig. 3K), while its level was reduced after treatment with tilianin. The above findings suggested that the protection of tilianin on HFHC-fed mice may be partly attributed to alleviating oxidative stress.

Tilianin Inhibited Hepatic Inflammation and the Activation of NLRP3 Inflammasome in HFHC-Fed Mice

Inflammation is a pivotal driver in the initiation and progression of NAFLD.²⁰⁾ Results of clinical samples showed higher serum IL-18 and IL-1β levels in obese children with NAFLD compared with normal children (Supplementary Figs. S1A, B), indicating increased levels of inflammation in obese children with NAFLD. In mouse model, hepatic expression of F4/80 was increased in HFHC-fed mice, indicating an exaggerated macrophage infiltration (Fig. 4A). Administration of tilianin to HFHC-fed mice reversed such induction. We further analyzed the serum levels of pro-inflammatory cytokines in mice. HFHC feeding elevated serum TNF-α, IL-1β, IL-6 and IL-18 levels (Figs. 4B–E). Tilianin administration reduced serum levels of these four pro-inflammatory cytokines in HFHC-fed mice. Consistent with changes in serum levels of pro-inflammatory cytokines, tilianin suppressed the elevated hepatic levels of TNF-α, IL-1β and IL-18 in HFHC-fed mice (Figs. 4F–H). Given that NLRP3 inflammasome is closely associated with the secretion and activation of pro-inflammatory cytokines,¹⁰⁾ the protein levels of inflammasome components NLRP3, pro-caspase-1, active (cleaved) caspase-1 were examined by Western blotting. HFHC fed-mice had augmented expression of active caspase-1 and NLRP3 (Figs. 4I–L), while mice treated with tilianin decreased these protein levels. These findings supported that tilianin was able to reduce HFHC-induced hepatic inflammation and inhibited the activation of NLRP3 inflammasome.

Fig. 4. Tilianin Inhibited Hepatic Inflammatory Response and the Activation of NLRP3 Inflammasome in HFHC-Fed Mice

(A) F4/80 immunofluorescence analysis in the liver. (B–E) Serum levels of TNF-α, IL-1β, IL-6, and IL-18 were determined by ELISA. (F–H) ELISA analysis of TNF-α, IL-1β and IL-18 levels in mice liver. (I–L) Pro caspase-1, active caspase-1 and NLRP3 protein levels in liver were determined by Western blot.

DISCUSSION

In recent decades, obesity in children has been a global public health problem, and its prevalence has surpassed that of adults. Obesity is a major risk factor for childhood NAFLD.²¹⁾ With different lifestyles than adults, such as minimal alcohol and drug intake, pediatric NAFLD have some special pathological manifestations.²²⁾ In this study, children with NAFLD had higher values of BMI, waistline, ALT, AST, GGT, TC, LDL-C, IL-18, and IL-1β compared to normal children. To more closely approximate the pathological state of NAFLD in obese children, 21-d-old mice fed with HFHC diet were used in this study. Excessive intake of HFHC is a main cause of over accumulation of lipids in liver, which leads to NAFLD.²³⁾ In our models, H&E and Oil Red O pathological pictures showed that the HFHC-fed mice displayed liver typical lesions of NAFLD, including hepatocellular ballooning, inflammatory infiltration and steatosis. The abnormal levels of ALT, AST, GGT, HDL-C, LDL-C and TC in HFHC-fed mice also indicated the occurrence of hepatocyte injury caused by HFHC diet. In addition, the mouse model was consistent with the characteristics of children with NAFLD. These results supported the establishment of NAFLD in the mouse model. In spite of the therapeutic effect and mechanism of tilianin have been extensively investigated in various disorders owing to its various physiological activities,^13,14) its regulatory effect on NAFLD and the possible mechanisms are still unclear. In HFHC-fed mice, we found that administration of tilianin alleviated the above pathological damage, and improved lipid metabolism, oxidative stress and inflammation. Our study demonstrated that the activation of NLRP3 inflammasome was suppressed by tilianin, it contributed to the reduction of liver inflammation in NAFLD.⁸⁾

Impaired hepatic function caused excess accumulation of lipids leads to hepatocyte steatosis, which is a predisposing factor and distinctive feature of NAFLD.²⁴⁾ Various metabolic enzymes and molecules are involved in the pathological development of hepatic fat deposition in NAFLD. As markers of hepatic steatosis, we detected abnormal levels of HDL-C, LDL-C and TC in HFHC-fed mice. Furthermore, the high protein levels of ACC1, FAS, SREBP-1c and LXRα were found in HFHC-fed mice. This finding was also confirmed in a clinical research.²⁵⁾ SREBP-1c is elevated in experimental mouse model with excess fat, it may be a staple factor in the mechanisms of obesity-related liver disease.²⁶⁾ As a nuclear transcription factor, SREBP-1c regulates the expression of multiple enzymes related to fatty acids, phospholipids, cholesterol, triglycerides by activating transcription.²⁷⁾ FAS and ACC1 are responsible for de novo lipogenesis, and are transcriptionally upregulated by SREBP-1c.²⁸⁾ ACC1 catalyzes malonyl-CoA production to limit the speed of fatty acid synthesis.²⁹⁾ A clinical study showed that LXRα expression increased with the progression of NAFLD, and was positively correlated with liver fat accumulation and inflammation.³⁰⁾ The lipid metabolism regulatory effect of LXRα in NAFLD was mediated by binding to the SREBP-1c promoter to transcriptionally regulate SREBP-1c expression.^25,31) Tilianin exerted an anti-hyperlipidemic effect and reduced serum triglyceride and TC levels in diabetic rats,¹³⁾ suggesting its potential of regulate lipid metabolism. In current study, administration of tilianin reversed the abnormal levels of HLD-C, LDL-C and TC in HFHC-fed mice and reduced hepatic lipid accumulation by suppressing the expression of proteins associated with hepatic lipogenesis.

Growing scientific evidences support oxidative stress as a pivotal mechanism in the pathogenesis of NAFLD.^32,33) Oxidative and antioxidant defense systems maintain homeostasis in healthy liver tissues. The overproduction of ROS caused by abnormal ROS production or scavenging mechanisms disrupts the oxidative-antioxidant balance in the liver, triggers oxidative stress, and forms lipid peroxides, such as 4-HNE.³³⁾ Excess ROS and lipid peroxides subsequently lead to hepatocellular damage and contribute to NAFLD progression.³⁴⁾ In our mouse model, the production of oxidative stress was detected in liver sections. Studies by other researchers indicated that tilianin alleviated oxidative stress through reducing ROS and lipid peroxidation products, increasing the activity of antioxidants.^14,35) Similarly, our results revealed that tilianin exhibited an antioxidative effect in NAFLD, as evidenced by reduced overproduction of ROS and 4-HNE, thereby relieving hepatocyte damage.

Inflammation is considered to be one of the hallmarks of the progression of NAFLD, contributing to the deterioration of simple fatty deposits (steatosis) into NASH.²⁰⁾ Herein, we found that inflammatory cytokine levels were significantly increased in HFHC-fed mice. The production of these inflammatory cytokines was decreased by tilianin, suggesting an anti-inflammatory effect on NAFLD as previously reported.¹³⁾ The secretion of inflammatory cytokines leads to the aggregation of inflammatory cells, and ultimately exacerbates NAFLD.³⁶⁾ Recently, studies on NLRP3 inflammasome in disorders related to oxidative stress and inflammation including NAFLD has attracted considerable attention.³⁷⁾ In response to pathogen-related and injury-related molecular patterns, the NLRP3 inflammasome aggregates and activates.¹⁰⁾ Upon its activation, pro-caspase-1 is cleaved into activated caspase-1, and then IL-1β is matured and secreted, thereby inducing the expression of multiple inflammatory cytokines.⁸⁾ Activation of the NLRP3 inflammasome is multi-pathway, and ROS is considered to be one of the important molecular mechanisms. Intracellular ROS overproduction mediates the assembly and activation of the NLRP3 inflammasome by ROS-related pathways, subsequently triggering a series of inflammatory cascades.³⁸⁾ However, the association of ROS and NLRP3 activation in NAFLD mice requires further experiments to confirm. Additionally, tilianin restricted IL-6, IL-1β, TNF-α, NLRP3, caspase-1 expression, ROS production, improved inflammation and oxidative stress in diabetic rats.³⁹⁾ We observed increased NLRP3 and active caspase-1 in NAFLD mice. However, administration of tilianin caused a block in NLRP3 inflammasome activation, indicating tilianin can ameliorate hepatic inflammatory response and exert an anti-NAFLD effect.

Overall, we provide evidences that tilianin ameliorates lipid accumulation, oxidative stress, and inflammation in the liver of NAFLD obese mice, may partly through inhibiting NLRP3 inflammasome activation. These findings may facilitate the discovery of novel therapeutic strategies for NAFLD in obese children.

Acknowledgments

This research was funded by Anhui Medical University Scientific Research Fund Project (No. 2021xkj179).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) The Lancet Diabetes Endocrinology. Childhood obesity: a growing pandemic. The Lancet Diabetes & Endocrinology, 10, 1 (2022).
• 2) Smith JD, Fu E, Kobayashi MA. Prevention and management of childhood obesity and its psychological and health comorbidities. Annu. Rev. Clin. Psychol., 16, 351–378 (2020).
• 3) Shimizu K, Ono M, Imoto A, Nagayama H, Tetsumura N, Terada T, Tomita K, Nishinaka T. Cranberry attenuates progression of non-alcoholic fatty liver disease induced by high-fat diet in mice. Biol. Pharm. Bull., 42, 1295–1302 (2019).
• 4) Geurtsen ML, Jaddoe VWV, Salas LA, Santos S, Felix JF. Newborn and childhood differential DNA methylation and liver fat in school-age children. Clinical Epigenetics, 12, 3 (2020).
• 5) Li H, Ying H, Hu A, Hu Y, Li D. Therapeutic effect of gypenosides on nonalcoholic steatohepatitis via regulating hepatic lipogenesis and fatty acid oxidation. Biol. Pharm. Bull., 40, 650–657 (2017).
• 6) Gao H, Zeng Z, Zhang H, Zhou X, Guan L, Deng W, Xu L. The glucagon-like peptide-1 analogue liraglutide inhibits oxidative stress and inflammatory response in the liver of rats with diet-induced non-alcoholic fatty liver disease. Biol. Pharm. Bull., 38, 694–702 (2015).
• 7) Ishikawa H, Ino S, Nakashima T, Matsuo H, Takahashi Y, Kohda C, Ōmura S, Tanaka K. Improvement effects of trehangelin A on high-fat diet causing metabolic clinical conditions. Biol. Pharm. Bull., 42, 2095–2101 (2019).
• 8) Mridha AR, Wree A, Robertson AAB, Yeh MM, Johnson CD, Van Rooyen DM, Haczeyni F, Teoh NCH, Savard C, Ioannou GN, Masters SL, Schroder K, Cooper MA, Feldstein AE, Farrell GC. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J. Hepatol., 66, 1037–1046 (2017).
• 9) Gaul S, Leszczynska A, Alegre F, Kaufmann B, Johnson CD, Adams LA, Wree A, Damm G, Seehofer D, Calvente CJ, Povero D, Kisseleva T, Eguchi A, McGeough MD, Hoffman HM, Pelegrin P, Laufs U, Feldstein AE. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. J. Hepatol., 74, 156–167 (2021).
• 10) Mangan MSJ, Olhava EJ, Roush WR, Seidel HM, Glick GD, Latz E. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat. Rev. Drug Discov., 17, 588–606 (2018).
• 11) Wan X, Xu C, Yu C, Li Y. Role of NLRP3 inflammasome in the progression of NAFLD to NASH. Can. J. Gastroenterol. Hepatol., 2016, 6489012 (2016).
• 12) Akanda MR, Uddin MN, Kim I-S, Ahn D, Tae H-J, Park B-Y. The biological and pharmacological roles of polyphenol flavonoid tilianin. Eur. J. Pharmacol., 842, 291–297 (2019).
• 13) García-Díaz JA, Navarrete-Vázquez G, García-Jiménez S, Hidalgo-Figueroa S, Almanza-Pérez JC, Alarcón-Aguilar FJ, Gómez-Zamudio J, Cruz M, Ibarra-Barajas M, Estrada-Soto S. Antidiabetic, antihyperlipidemic and anti-inflammatory effects of tilianin in streptozotocin-nicotinamide diabetic rats. Biomed. Pharmacother., 83, 667–675 (2016).
• 14) Tian L, Cao W, Yue R, Yuan Y, Guo X, Qin D, Xing J, Wang X. Pretreatment with Tilianin improves mitochondrial energy metabolism and oxidative stress in rats with myocardial ischemia/reperfusion injury via AMPK/SIRT1/PGC-1 alpha signaling pathway. J. Pharmacol. Sci., 139, 352–360 (2019).
• 15) Liou C-J, Wu S-J, Shen S-C, Chen L-C, Chen Y-L, Huang W-C. Acacetin protects against non-alcoholic fatty liver disease by regulating lipid accumulation and inflammation in mice. Int. J. Mol. Sci., 23, 4687 (2022).
• 16) Kohli R, Kirby M, Xanthakos SA, Softic S, Feldstein AE, Saxena V, Tang PH, Miles L, Miles MV, Balistreri WF, Woods SC, Seeley RJ. High-fructose, medium chain trans fat diet induces liver fibrosis and elevates plasma coenzyme Q9 in a novel murine model of obesity and nonalcoholic steatohepatitis. Hepatology, 52, 934–944 (2010).
• 17) Kim S-H, Hong J-H, Yang W-K, Geum J-H, Kim H-R, Choi S-Y, Kang Y-M, An H-J, Lee Y-C. Herbal combinational medication of Glycyrrhiza glabra, Agastache rugosa containing glycyrrhizic acid, tilianin inhibits neutrophilic lung inflammation by affecting CXCL2, interleukin-17/STAT3 Signal Pathways in a Murine Model of COPD. Nutrients, 12, 926 (2020).
• 18) Wang L, Chen Q, Zhu L, Zeng X, Li Q, Hu M, Wang X, Liu Z. Simultaneous determination of tilianin and its metabolites in mice using ultra-high-performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study. Biomed. Chromatogr., 32, e4139 (2018).
• 19) Dang Y, Xu J, Zhu M, Zhou W, Zhang L, Ji G. Gan-Jiang-Ling-Zhu decoction alleviates hepatic steatosis in rats by the miR-138-5p/CPT1B axis. Biomed. Pharmacother., 127, 110127 (2020).
• 20) Arrese M, Cabrera D, Kalergis AM, Feldstein AE. Innate immunity and inflammation in NAFLD/NASH. Dig. Dis. Sci., 61, 1294–1303 (2016).
• 21) Lee EY, Yoon K-H. Epidemic obesity in children and adolescents: risk factors and prevention. Front. Med., 12, 658–666 (2018).
• 22) Schwimmer JB, Behling C, Newbury R, Deutsch R, Nievergelt C, Schork NJ, Lavine JE. Histopathology of pediatric nonalcoholic fatty liver disease. Hepatology, 42, 641–649 (2005).
• 23) Zhou X, Fouda S, Li D, Zhang K, Ye J-M. Involvement of the autophagy-ER stress axis in high fat/carbohydrate diet-induced nonalcoholic fatty liver disease. Nutrients, 12, 2626 (2020).
• 24) Tariq Z, Green CJ, Hodson L. Are oxidative stress mechanisms the common denominator in the progression from hepatic steatosis towards non-alcoholic steatohepatitis (NASH)? Liver Int., 34, e180–e190 (2014).
• 25) Higuchi N, Kato M, Shundo Y, Tajiri H, Tanaka M, Yamashita N, Kohjima M, Kotoh K, Nakamuta M, Takayanagi R, Enjoji M. Liver X receptor in cooperation with SREBP-1c is a major lipid synthesis regulator in nonalcoholic fatty liver disease. Hepatol. Res., 38, 1122–1129 (2008).
• 26) Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol. Cell, 6, 77–86 (2000).
• 27) Foufelle F, Ferré P. New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c. Biochem. J., 366, 377–391 (2002).
• 28) Zhao W-J, Bian Y-P, Wang Q-H, Yin F, Yin L, Zhang Y-L, Liu J-H. Blueberry-derived exosomes-like nanoparticles ameliorate nonalcoholic fatty liver disease by attenuating mitochondrial oxidative stress. Acta Pharmacol. Sin., 43, 645–658 (2022).
• 29) Bao X, Koorengevel MC, Groot Koerkamp MJA, Homavar A, Weijn A, Crielaard S, Renne MF, Lorent JH, Geerts WJ, Surma MA, Mari M, Holstege FCP, Klose C, de Kroon AIPM. Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast. EMBO J., 40, e107966 (2021).
• 30) Ahn SB, Jang K, Jun DW, Lee BH, Shin KJ. Expression of liver X receptor correlates with intrahepatic inflammation and fibrosis in patients with nonalcoholic fatty liver disease. Dig. Dis. Sci., 59, 2975–2982 (2014).
• 31) Yoshikawa T, Shimano H, Amemiya-Kudo M, Yahagi N, Hasty AH, Matsuzaka T, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Osuga J, Harada K, Gotoda T, Kimura S, Ishibashi S, Yamada N. Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter. Mol. Cell. Biol., 21, 2991–3000 (2001).
• 32) Farzanegi P, Dana A, Ebrahimpoor Z, Asadi M, Azarbayjani MA. Mechanisms of beneficial effects of exercise training on non-alcoholic fatty liver disease (NAFLD): roles of oxidative stress and inflammation. Eur. J. Sport Sci., 19, 994–1003 (2019).
• 33) Chen Z, Tian R, She Z, Cai J, Li H. Role of oxidative stress in the pathogenesis of nonalcoholic fatty liver disease. Free Radic. Biol. Med., 152, 116–141 (2020).
• 34) Yang J, Fernández-Galilea M, Martínez-Fernández L, González-Muniesa P, Pérez-Chávez A, Martínez JA, Moreno-Aliaga MJ. Oxidative stress and non-alcoholic fatty liver disease: effects of omega-3 fatty acid supplementation. Nutrients, 11, 872 (2019).
• 35) Wang Y, Yuan Y, Wang X, Wang Y, Cheng J, Tian L, Guo X, Qin D, Cao W. Tilianin post-conditioning attenuates myocardial ischemia/reperfusion injury via mitochondrial protection and inhibition of apoptosis. Med. Sci. Monit., 23, 4490–4499 (2017).
• 36) Zhu H, Zhang Q, Chen G. CXCR6 deficiency ameliorates ischemia-reperfusion injury by reducing the recruitment and cytokine production of hepatic NKT cells in a mouse model of non-alcoholic fatty liver disease. Int. Immunopharmacol., 72, 224–234 (2019).
• 37) Xu Y, Yang C, Zhang S, Li J, Xiao Q, Huang W. Ginsenoside Rg1 protects against non-alcoholic fatty liver disease by ameliorating lipid peroxidation, endoplasmic reticulum stress, and inflammasome activation. Biol. Pharm. Bull., 41, 1638–1644 (2018).
• 38) Qiu Z, He Y, Ming H, Lei S, Leng Y, Xia Z-Y. Lipopolysaccharide (LPS) aggravates high glucose- and hypoxia/reoxygenation-induced injury through activating ROS-dependent NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. J. Diabetes Res., 2019, 8151836 (2019).
• 39) Yao J, Li Y, Jin Y, Chen Y, Tian L, He W. Synergistic cardioptotection by tilianin and syringin in diabetic cardiomyopathy involves interaction of TLR4/NF-κB/NLRP3 and PGC1a/SIRT3 pathways. Int. Immunopharmacol., 96, 107728 (2021).