Vinpocetine represses the progression of nonalcoholic steatohepatitis in mice by mediating inflammasome components via NF-κB signaling

Abstract

Background

Inflammasome activation is known to be involved in nonalcoholic steatohepatitis (NASH). Vinpocetine is a derivative of vincamine and is reported to suppress the activation of inflammasome.

Methods

This study explored the therapeutical potential of Vinpocetine on NASH. Mice were fed with a choline-deficient (MCD) or chow diet in the presence or absence of Vinpocetine for 8 weeks. H&E staining and biochemical assays were determined to evaluate the hepatic steatosis and fibrosis symptoms. In addition, primary hepatocytes and Kupffer cells were isolated and induced by MCD or lipopolysaccharides/cholesterol crystals with or without Vinpocetine. ELISAs, qPCR, and Western blotting were applied to determine the levels of NASH-related biomarkers in both in vivo mouse model and in vitro cell models.

Results

Treatment of Vinpocetine did not cause observable side effects against and MCD-induced cells and mouse NASH model. However, treatment of Vinpocetine ameliorated hepatic steatosis and fibrosis and suppressed the levels of alanine transaminase and aspartate transferase in the mouse NASH model. In addition, treatment of Vinpocetine suppressed the mRNA and protein levels of inflammasome components both in vitro and in vivo.

Conclusion

Vinpocetine suppressed NASH in mice by mediating inflammasome components via nuclear factor κB signaling.

Resumen

Antecedentes

Se sabe que la activación del inflamasoma está implicada en la esteatohepatitis no alcohólica (EHNA). La vinpocetina es un derivado de la vincamina que, según los informes, suprime la activación del inflamasoma.

Métodos

Este estudio exploró el potencial terapéutico de la vinpocetina en la EHNA. Durante 8 semanas se alimentó a ratones con una dieta deficiente en colina (MCD) o con una dieta chow en presencia o ausencia de vinpocetina. Se realizaron tinciones de H&E y ensayos bioquímicos para evaluar los síntomas de esteatosis hepática y fibrosis. Además, se aislaron hepatocitos primarios y células de Kupffer y se indujeron mediante MCD o cristales de lipopolisacáridos/colesterol con o sin vinpocetina. Se aplicaron ELISA, qPCR y Western blotting para determinar los niveles de biomarcadores relacionados con la EHNA tanto en el modelo de ratón in vivo como en los modelos celulares in vitro.

Resultados

El tratamiento con vinpocetina no causó efectos secundarios observables contra las células y el modelo de ratón de EHNA inducidos por MCD. Sin embargo, el tratamiento con vinpocetina mejoró la esteatosis hepática y la fibrosis y suprimió los niveles de alanina transaminasa y de aspartato transferasa en el modelo de EHNA de ratón. Además, el tratamiento con vinpocetina suprimió los niveles de ARNm y proteínas de los componentes del inflamasoma tanto in vitro como in vivo.

Conclusiones

La vinpocetina suprimió la EHNA en ratones por mediación de los componentes del inflamasoma a través de la señalización del factor nuclear κB.

Introduction

The prevalence of nonalcoholic fatty liver disease (NAFLD) is around 25% global and increasing annually.¹ In the late stage of NAFLD, the involvement of other factors including oxidative stress and inflammation accelerates the development of NAFLD.² Nonalcoholic steatohepatitis (NASH) is an inflammation condition in the liver.^3,4 However, the pathogenesis of NASH is complex and still not fully understood. Currently, multiple parallel hits theory, other than ‘two hits’, is well accepted in the pathogenesis of NASH.⁴ In brief, lipid accumulation is regarded as an early sign for fatty liver disease.^5,6 The nonesterified fatty acid is formed as hepatic triglyceride-enriched lipid droplets, which lead to liver steatosis. Lipotoxicity also activates the innate immune system, leading to the trafficking of immune cells, the activation of inflammasome components, and the production of pro-inflammatory cytokines and chemokines.^5–7 As a result, these multi-factors result in the development of NAFLD disease into NASH.^5,7

NOD-, LRR- and pyrin domain-containing protein 3 (NRLP3) inflammasome is well known to regulate the innate immune response.^8,9 The upregulation of NLRP3 inflammasome is frequently observed in a series of inflammation-mediated diseases including neurodegenerative disease, atherosclerosis, inflammatory bowel disease, rheumatoid arthritis, and NASH.^9–12 More recently, the roles of NRLP3 inflammasome activation in the development of NASH have drawn much attention from scientists.^13–15 As a multiprotein complex, inflammasome is consists of sensor proteins, adaptor proteins, and precursors.¹⁴ The activation of NLRP3 requires two signals including signal 1 and signal 2.¹⁰ Signal 1 inducer triggers toll-like receptor-mediated signaling cascades and the productions of downstream biomarkers including pro-interleukin (IL)-1β and pro-IL-18.¹³ Signal 2 inducers mediated the assembly of the inflammasome. Pro-IL-1β and pro-IL-18 were cleaved by the matured Caspase-1, becoming IL-1β and IL-18, which play biological functions in NASH.¹⁵ In the development of NASH, hepatocyte steatosis provides a series of stimuli (cholesterol crystal and ATP, etc.), which triggers the activation of NRLP3 inflammasome.^13,15 Interestingly, a previous study reported that the blockage of NLRP3 inflammasome suppresses liver inflammation and fibrosis in NASH in murine models.¹⁶ Therefore, it is worthwhile to develop novel strategies against NLRP3 inflammasome activation for NASH therapy.

Vinpocetine is a derivative of vincamine found in Voacanga africana and exerts a diverse biological activity in cerebrovascular diseases, acute ischemic stroke, and atherosclerosis.^17,18 In addition, Vinpocetine is also reported to have anti-inflammation activities in a series of pre-clinical and clinical studies.^18,19 In 2021, Han and colleagues reported that treatment with Vinpocetine attenuates ischemic stroke by the inhibition of NLRP3 inflammasome and NF-κB signaling pathways.²⁰ However, it is still unknown whether Vinpocetine exerts beneficial effects against NASH. Herein, this study aims to explore the therapeutic potential of Vinpocetine on NASH and the underlying mechanisms.

Material and methods

Animals and experimental design

Thirty-five C57BL/6 mice were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animal studies were approved by Jiangnan University Medical Center. To construct the NASH mouse model, the mice were fed with a choline-deficient (MCD) diet for 8 weeks. Besides, the mice fed with chow diet were used as the control group.

In this study, the animals were divided into 5 groups (n = 7). In group 1, the mice fed with the chow diet were intraperitoneally injected with Vinpocetine (Vinp, 20 mg/kg every two days) for 8 weeks. In group 2, the mice fed with the chow diet were intraperitoneally injected with the same volume of vehicle for 8 weeks. In group 3, the mice fed with the MCD diet were intraperitoneally injected with Vinpocetine (20 mg/kg every two days) for 8 weeks. In group 4, the mice fed with the MCD diet were intraperitoneally injected with Vinpocetine (10 mg/kg every two days) for 8 weeks. In group 5, the mice fed with the MCD diet were intraperitoneally injected with the same volume of vehicle for 8 weeks. After 8 weeks, the mice were utilized and the plasma and liver tissues were collected for further biochemical analysis.

Isolation of primary hepatocytes and treatment

Mice primary hepatocytes were isolated by using the collagenase perfusion method according to a previous reported method.²¹ The isolated primary hepatocytes were seeded on collagen-coated plates and incubated with William's E Medium supplement with 1% bovine serum albumin, 1% Peni-strep solution, nicotinamide (10 mM), and HEPES Buffer Solution (10 mM). The cells were then incubated with the MCD medium with Vinpocetine (50 μM) or vehicle for another 24 h. The supernatant was collected for biochemical assays and the cell lysate was prepared for qPCR and Western blotting analysis, respectively.

Isolation of primary Kupffer cells and treatment

Mice primary Kupffer cells were isolated as previously reported.²² In brief, liver was sequentially perfused with buffer A and buffer B. The perfused liver tissues were excised and shaken in medium containing collagenase IV followed by incubating with medium containing 2% fetal bovine serum. Cell suspension was centrifuged and washed. Cell pellets were then resuspended and layered on a double Percoll gradient (25% and 50%). Flow cytometry was used to analyze the cell types. Cholesterol crystals were prepared as Samstad described.²³

Western blotting

The liver tissues or primary hepatocytes were collected and homogenized as the tissue lysate by using a cold RIPA buffer containing protease inhibitor. After the centrifugation at 12,000 × g for 10 min, the tissue supernatant was collected and qualified by using the BCA reagent. The protein samples were then loaded on Precast Gels followed by the transferring on the PVDF membrane. Next, the membrane was incubated 5% bovine serum albumin at room temperature for 2 h. Next, primary antibodies against Caspase 1 (Cell Signaling Technology, Danvers, MA), Caspase 11 (Cell Signaling Technology), Pro-IL-1β (Cell Signaling Technology), p-IκBα (Cell Signaling Technology), Timd4 (Thermo Fisher, Waltham, MA), Clec4F (Thermo Fisher) and β-actin (Proteintech, Wuhan, Hubei, China) were incubated with membrane overnight at 4 °C. The membrane was then blotted with the secondary antibodies prior to the chemiluminescent reaction.

qPCR

Total RNAs were extracted from the liver tissues or primary hepatocytes by using Trizol reagent. Next, a reverse transcription reaction was performed. The sequences of primers were listed as follows. GAPDH forward: 5′-AGG TCG GTG TGA ACG GAT TTG-3′, and reverse: 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′; Caspase-1 forward: 5′-ACA AGG CAC GGG ACC TAT G-3′, and reverse: 5′-TCC CAG TCA GTC CTG GAA ATG-3′, Caspase-11 forward: 5′-ACA AAC ACC CTG ACA AAC CAC-3′, and reverse: 5′-CAC TGC GTT CAG CAT TGT TAA A-3′; IL-1β forward: 5′-GCA ACT GTT CCT GAA CTC AAC T-3′, and reverse: 5′-ATC TTT TGG GGT CCG TCA ACT-3′; TGF-β1 forward: 5′-CTC CCG TGG CTT CTA GTG C-3′, and reverse: 5′-GCC TTA GTT TGG ACA GGA TCT G-3′; α-SMA forward: 5′-GTC CCA GAC ATC AGG GAG TAA-3′; and reverse: 5′-TCG GAT ACT TCA GCG TCA GGA-3′; COL1-a1 forward: 5′-GCT CCT CTT AGG GGC CAC T-3′ and reverse: 5′-CCA CGT CTC ACC ATT GGG G-3′; COL3-a1 forward: 5′-TGC TGG AAA GGA TGG AGA GT-3′, and reverse: 5′-TGG GCC TTT GAT ACC TGG AG-3′; TIMP-1 forward: 5′-CAT GGA AAG CCT CTG TGG AT-3′, and reverse: 5′-CTC AGA GTA CGC CAG GGA AC-3′; iNOS forward: 5′-GGA GTG ACG GCA AAC ATG ACT-3′, and reverse: 5′-TCG ATG CAC AAC TGG GTG AAC-3′; Arg1 forward: 5′-CTC CAA GCC AAA GTC CTT AGA G-3′, and reverse: 5′-AGG AGC TGT CAT TAG GGA CAT C-3′; Cd206 forward: 5′-CTC TGT TCA GCT ATT GGA CGC-3′, and reverse: 5′-CGG AAT TTC TGG GAT TCA GCT TC-3′; Cd11c forward: 5′-CTG GAT AGC CTT TCT TCT GCT G-3′, and reverse: 5′-GCA CAC TGT GTC CGA ACT CA-3′; Nlrp3 forward: 5′-ATT ACC CGC CCG AGA AAG G-3′, and reverse: 5′-TCG CAG CAA AGA TCC ACA CAG-3′; Asc forward: 5′-CTT GTC AGG GGA TGA ACT CAA AA-3′, and reverse: 5′-GCC ATA CGA CTC CAG ATA GTA GC-3′. To ensure the accuracy of the reaction, the Melt curves were applied. The relative mRNA expression levels of target genes were normalized to the internal control GAPDH.

Liver histopathological examination

The liver tissues were collected and fixed with paraformaldehyde solutions (4%). Hematoxylin and eosin (H&E) staining was applied to evaluate the histopathological change of the liver tissues. Blinded histological analysis was performed by a pathologist. Steatosis and ballooning score was then determined as previously described.²⁴ Steatosis score is scaled from 0 to 4. 0 indicates that the steatosis area is less than 5%. 1 indicates that the steatosis area is between 5% and 25%. 2 indicates that the steatosis area is between 25% and 50%. 3 indicates that the steatosis area is between 50% and 75%. 4 indicates that steatosis area is between 75% and 100%. The ballooning score is scaled from 0 to 3. 0 indicates no ballooning. 1 indicates mild ballooning. 2 indicates moderate ballooning. 3 indicates severe ballooning.

Biochemical assays

Alanine transaminase (ALT) and aspartate transferase (AST) from serum or medium were detected by using commercialized reagent. Liver tissue homogenization buffer was prepared and hepatic triglyceride and cholesterol were measured by using Triglycerides LiquiColor Test reagent (Interchim, Montluçon, France) and Cholesterol/Cholesteryl Ester Quantitation Kit (BioVision, Milpitas, CA, USA), respectively. Hepatic lipid peroxidation was determined by thiobarbituric acid reactive substances (TBARS) assay (Sigma, St Louis, MO). Hepatic hydroxyproline was determined by previously reported method.²⁴

Flow cytometry

Leukocytes were isolated from liver tissues as previously reported.²¹ After the tissue lysate was prepared, the tissue extract was filtered through a 70 μm cell strainer followed by the staining with APC-conjugated F4/80 and PE-Cy7 conjugated CD11c. The cell suspension was determined by using a flow cytometer (BD Biosciences, Heidelberg, Germany) and the frequency of cell populations was determined by using FlowJo Software (Ashland, OR, United States).

ELISAs

Tissue lysate was prepared and protein concentrations were qualified by using BCA assay. Next, cytokine panels including IL-1β, IL-6, TNF-α, and CCL-2 in the liver tissues were determined by using specific ELISAs, according to the manufacturers’ protocols (R&D Systems, Minneapolis, MN).

Data analysis

In this study, two-way ANOVA analysis was performed by using GraphPad Prism 7. Data were displayed as the means ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, indicates significant difference between the two groups. ns indicates that no significance was observed between two groups. nd indicates the value that was not detected.

Results

Treatment of Vinpocetine attenuated hepatic steatosis in NASH mice

We evaluated the effects of Vinpocetine on MCD-induced hepatic steatosis. As shown in Fig. 1A, B, treatment of Vinpocetine did not exert toxic effects on liver tissues. There is no significance for steatosis and ballooning score between the Vinpocetine-treated control group and the control group. Interestingly, treatment of Vinpocetine (10 and 20 mg/kg) significantly reduced steatosis and ballooning score when compared with those in the MCD-induced vehicle groups (Fig. 1A–C).

Figure 1.

Treatment of Vinpocetine (Vinp) attenuated MCD-induced hepatic steatosis in mice. Mice were exposed to methionine–choline deficient (MCD) or chow diet with the treatment of Vinpocetine (10 or 20 mg/kg) or vehicle for 8 weeks. (A–C) Hepatic steatosis and ballooning scores were calculated. Besides, (D–F) liver triglyceride, serum ALT level, serum AST level, and liver triglyceride were detected by using commercialized kits (n = 7 per group).

We did not observe a significant difference for hepatic triglyceride, serum ALT, and serum AST between the Vinpocetine-treated control group and the control group (Fig. 1D–F). However, treatment of Vinpocetine (10 and 20 mg/kg) decreased hepatic triglyceride, serum ALT, and serum AST when compared with those in the MCD-induced vehicle groups. Moreover, our results showed that treatment of Vinpocetine (20 mg/kg) reduced hepatic cholesterol and lipid peroxide (Supplemental material Fig. S1A and S1B). Our results indicated the ameliorated effects of Vinpocetine on MCD-induced hepatic steatosis.

Treatment of Vinpocetine attenuated hepatic fibrosis in NASH mice

We also determined whether the presence of Vinpocetine affected the mRNA levels of hepatic fibrosis-related biomarkers. We observed that the presence of Vinpocetine did not affect the mRNA levels of Tgfb1, a-Sma, Timp1, Col1a1, and Col3a1 in the liver tissues of the mice fed with chow diet (Fig. 2A–E). As expected, those biomarkers were significantly elevated in the mice fed with the MCD diet. However, the presence of Vinpocetine (10 and 20 mg/kg) dramatically reduced the mRNA levels of Tgfb1, a-Sma, Timp1, Col1a1, and Col3a1 in the liver tissues (Fig. 2A–E). In addition, we also found that treatment of Vinpocetine (20 mg/kg) significantly reduced hydroxyproline content induced by MCD (Fig. S1C). Taken together, these results supported that treatment of Vinpocetine attenuated MCD-induced hepatic fibrosis in mice.

Figure 2.

Treatment of Vinpocetine attenuated MCD-induced hepatic fibrosis in mice. Mice were exposed to MCD or chow diet with the treatment of Vinpocetine (20 mg/kg) or vehicle for 8 weeks. (A–E) At the end of the experimental period, the mRNA expression levels of Tgfb1, a-Sma, Timp1, Col1a1, and Col3a1 in the liver were determined by RT-qPCR (n = 7 per group).

Treatment of Vinpocetine inhibited inflammation in the liver in NASH mice

We next detected the frequency of macrophages and inflammation cytokines in the MCD-induced mouse model. We found that treatment of Vinpocetine did not significantly affect the population of CD11b⁺F4/80⁺ macrophages in the control group. However, treatment of Vinpocetine effectively reduced the population of CD11b⁺F4/80⁺ macrophages in the mice fed with the MCD diet (Fig. 3A). Moreover, we also observed that treatment of Vinpocetine did not affect the protein levels of IL-1β (Fig. 3B), IL-6 (Fig. 3C), TNF-α (Fig. 3D), and CCL-2 (Fig. 3E) in the liver in the control group, whereas treatment of Vinpocetine reduced the protein levels of those cytokines in the MCD-induced group. We detected IL-1β, IL-6, TNF-α and CCL2 in plasma of mice, and found that significant reduction upon Vinpocetine treatment (Fig. S2A–S2D). Moreover, we determined biomarkers including the M1 macrophage marker (iNOS), M2 macrophage marker (Arg1 and Cd206), and dendritic cell marker (Cd11c). Interestingly, we found that the levels of iNOS, Arg1, Cd206, and Cd11c were significantly decreased in the Vinp-treated groups (Fig. S2E–S2H), indicating that Vinpocetine did not affect macrophage polarization. The protein levels of tissue resident macrophages markers Timd4, Clec4F were detected, and it was found that Vinpocetine down-regulated the levels of Timd4 and Clec4F in liver (Fig. S2I–S2K). These results suggested the anti-inflammatory effects of Vinpocetine on the MCD-induced mouse model.

Figure 3.

Treatment of Vinpocetine inhibited MCD-induced inflammation in the liver. Mice were exposed to MCD or chow diet with the treatment of Vinpocetine (20 mg/kg) or vehicle for 8 weeks. (A) The infiltration of macrophages (CD11b⁺F4/80⁺) in the liver was determined by flow cytometer. (B–E) The protein expression levels of IL-1β, IL-6, TNF-α, and CCL-2 in the liver were detected by specific ELISAs (n = 7 per group).

Treatment of Vinpocetine downregulated MCD-induced inflammasome in mice

We also evaluated the effects of Vinpocetine on the regulation of inflammasome activation induced by MCD. It was firstly found that the mRNA levels of Nlrp3 and Asc in liver tissues of MCD-induced mice were significantly down-regulated by Vinpocetine treatment (Fig. S2L, S2M). Furthermore, both qPCR and Western blotting results showed that treatment of Vinpocetine did not affect the mRNA and protein levels of inflammasome-associated genes including Caspase 1, Caspase 11, and Il1b (Fig. 4). In addition, qPCR showed that treatment of Vinpocetine significantly reduced the mRNA levels of Caspase 1 (Fig. 4A), Caspase 11 (Fig. 4B), and Il1b (Fig. 4C) in liver tissues in the MCD-induced group. Western blotting showed that treatment of Vinpocetine significantly reduced the protein levels of cleaved Caspase-1 (Fig. 4F), pro-Caspase-11 (Fig. 4G), cleaved Caspase-11 (Fig. 4H), and mature IL-1β (Fig. 4J) in the MCD-induced group. Furthermore, we also found that treatment of Vinpocetine did not affect the protein levels of pro-Caspase-1 (Fig. 4E) and pro-IL-1β (Fig. 4I). Our data indicated the regulatory effects of Vinpocetine on the activation of the inflammasome.

Figure 4.

Treatment of Vinpocetine downregulated MCD-induced inflammasome in mice. Mice were exposed to MCD or chow diet with the treatment of Vinpocetine (20 mg/kg) or vehicle for 8 weeks. (A–C) The mRNA expression levels of Caspase 1, Caspase 11, and Il1b in the liver were determined by RT-qPCR. (D–J) The protein levels of Caspase-1, Caspase-11, and IL-1β in the liver were detected by using Western blotting (n = 7 per group).

Treatment of Vinpocetine decreased the expressions of inflammasome components

To confirm the results obtained from the in vivo studies, we also measured the expressions of inflammasome components in MCD-induced primary hepatocytes. Similarly, it was observed that the mRNA levels of Nlrp3 and Asc in the cultured primary hepatocytes treated by MCD medium were significantly down-regulated by Vinpocetine treatment (Fig. S2N, S2O). We did not observe a significant change in the mRNA and protein levels of inflammasome-associated genes in the primary hepatocytes treated with Vinpocetine (Fig. 5A–G). However, we found that treatment of Vinpocetine reduced the mRNA levels of Caspase 1 (Fig. 5A), Caspase 11 (Fig. 5B), and Il1b (Fig. 5C) in the MCD-induced hepatocytes. Consistently, treatment of Vinpocetine also reduced the protein levels of pro-Caspase-1 (Fig. 5E), pro-Caspase-11 (Fig. 5F), and pro-IL-1β (Fig. 5G). Moreover, by measuring the medium ALT and AST, we observed that treatment of Vinpocetine reduced the elevation of ALT (Fig. 5H) and AST (Fig. 5I) in the medium caused by the MCD.

Figure 5.

Treatment of Vinpocetine decreased the expressions of inflammasome components. Primary hepatocytes from mice were cultured and exposed to MCD medium with the treatment of Vinpocetine (50 μM) or vehicle for 24 h. (A–C) The mRNA expression levels of Caspase 1, Caspase 11, and Il1b were determined by RT-qPCR. (D–G) The protein expression levels of Caspase-1, Caspase-11, and IL-1β were detected by Western blotting. (H–I) The levels of ALT and AST in the medium were detected (n = 4 per group).

In addition, we explored the effects of Vinp on the regulation of inflammasome activation in Kupffer cells. Our results revealed that treatment of Vinp reduced the mRNA levels of Caspase 1 (Fig. S3A), Caspase 11 (Fig. S3B), and Il1b (Fig. S3C) in the lipopolysaccharides/cholesterol crystals induced Kupffer cells. Consistently, treatment of Vinpocetine also reduced the protein levels of p-IκBα (Fig. S3E), pro-Caspase-1 (Fig. S3F), cleaved Caspase-1 (Fig. S3G), pro-Caspase-11 (Fig. S3H), cleaved Caspase-11 (Fig. S3I), and pro-IL-1β (Fig. S3J), and IL-1β (Fig. S3K). Moreover, Kupffer conditioned medium from MCD mice up-regulated ALT and AST in hepatocytes (Fig. S3L, S3M), indicating that the cultured hepatocytes were damaged. However, Kupffer conditioned medium from MCD-Vinp mice could only weakly inhibit the increase of ALT, but failed to inhibit the increase of AST, indicating that Vinpocetine mainly directly acts on liver cells and Kupffer cells. These results suggested that Vinp attenuated inflammation in part by inhibiting NFκB signaling and inflammasome activation in Kupffer cells.

Treatment of Vinpocetine reduced the activation of NFκB signaling both in vivo and in vitro

We measured the protein expressions of NF-κB signaling protein in the MCD-induced mouse model and cell model. Interestingly, we observed that treatment of Vinpocetine decreased the phosphorylation of IκBα in the liver tissues of MCD-induced mice (Fig. 6A, B). Consistently, treatment of Vinpocetine decreased the phosphorylation of IκBα in the MCD-induced mouse liver hepatocytes (Fig. 6C, D).

Figure 6.

Treatment of Vinpocetine reduced the activation of NFκB signaling both in vivo and in vitro. Mice were exposed to MCD or chow diet with the treatment of Vinpocetine (20 mg/kg) or vehicle for 8 weeks. (A, B) The protein expression levels of p-IκBα in the liver were detected by Western blot (n = 7 mice per group). Primary hepatocytes from mice were cultured and exposed to the MCD medium with the treatment of Vinpocetineocetine (50 μM) or vehicle for 24 h. (C, D) The protein expression levels of p-IκBα were detected by Western blot (n = 4 per group).

Discussion

This study reported that Vinpocetine exerted therapeutic properties against the MCD-induced mouse NASH model and primary mouse hepatocytes. First, treatment of Vinpocetine ameliorated the pathological features of NASH including liver steatosis, the accumulation of liver triglyceride, and the elevation of serum ALT and AST. Second, treatment of Vinpocetine reduced the mRNA levels of hepatic fibrosis-related biomarkers. Third, treatment of Vinpocetine decreased the infiltration of macrophages and the release of pro-inflammatory cytokines. Moreover, treatment of Vinpocetine reduced the expressions of inflammasome components and the phosphorylation of IκBα in vitro and in vivo. More importantly, our results did not reveal observable effects in Vinpocetine-treated mice and cells. These results suggested that Vinpocetine might be a drug candidate for the treatment of NASH in clinical settings.

NASH is an inflammation condition in the liver, which is histologically featured by liver steatosis and fibrosis, liver cell ballooning, and inflammation.³ In addition to the histological change in the liver, two typical liver enzymes including ALT and AST are dramatically increased in most of the patients with NASH.^3,25 Another common feature of NASH is the accumulation of triglyceride in the hepatocytes.² To evaluate the therapeutic properties of Vinpocetine, we firstly constructed the MCD-induced mouse NASH model, which is a commonly used model in pre-clinical settings.^21,26 NASH mouse was characterized by a series of NASH-related histological changes including liver steatosis and ballooning, liver triglyceride accumulating, and the elevation of serum ALT and AST, which suggested the NASH model was successfully constructed. Interestingly, our results revealed that treatment of Vinpocetine effectively ameliorated the pathological features of NASH including liver steatosis, the accumulation of liver triglyceride, and the elevation of serum ALT and AST.

Liver fibrosis is another hallmark of NASH, which is triggered by lipotoxicity and inflammation.^5,27 A series of liver fibrogenesis and fibrinolysis biomarkers have been identified to predict and diagnose liver fibrosis.²⁷ For instance, transform growth factor-β is known as the most important cytokines in the induction of liver fibrosis by regulating macrophage activities, the activation of fibrogenic myofibroblasts, and epithelial mesenchymal transition.²⁸ The overexpression of alpha-smooth muscle actin (α-SMA) results in the production of extracellular matrix including different types of collagens.²⁹ Therefore, we further analyzed the mRNA levels of liver fibrosis biomarkers in the liver tissues with or without Vinpocetine. Our results showed that treatment of Vinpocetine significantly reduced the mRNA levels of Tgfb1, a-Sma, Timp1, Col1a1, and Col3a1, indicating that Vinpocetine might be beneficial for ameliorating liver fibrosis.

NASH is also featured by the inflammation response including the accumulation of macrophages and the release of pro-inflammatory cytokines in the liver tissues.^6,30 Macrophages are regarded as one the most important immune cells in the NASH, which are triggered by a series of stimuli such as cholesterol, fatty acid, and endotoxins.^30,31 The activation of macrophages initiates inflammation downstream cascade including the release of nitric oxide, pro-inflammatory cytokines, mediators, and cytokines.³¹ We also determined the infiltration of macrophages (CD11b⁺F4/80⁺) and the expressions of IL-1β, IL-6, TNF-α, and CCL-2. Our results showed that treatment of Vinpocetine effectively reduced the population of CD11b⁺F4/80⁺ macrophages and expressions of those cytokines and chemokine, suggesting the inhibitory effects of Vinpocetine on the inflammation. Interestingly, we did not observe that Vinpocetine affected macrophages polarization, as supported by the reduction of M1 macrophage marker (iNOS) and M2 macrophage marker (Cd206).³² However, further assays are needed to determine whether Vinp affected subtypes and populations of macrophages (M1 or M2 phenotype). Moreover, our results revealed that the inhibitory effects of Vinpocetine on the inflammation were associated with its inhibitory effects against the phosphorylation of IκBα.

The roles of the inflammasome in the NASH have drawn much attention in recent years.^13,15 Studies have revealed that the activation of the inflammasome is involved in the pathogenesis of NASH and the inhibition of inflammasome is able to effectively ameliorate the development of NASH in the mouse model.^13,16 Two different pathways including canonical and non-canonical pathways have been implicated to be involved in the activation of NLRP3 inflammasome.^14,33 Caspase-1 and Caspase-11 are key components in the canonical and non-canonical pathways, respectively.³³ In our study, we found that the canonical and non-canonical pathways are activated in the NASH model. Interestingly, in the MCD-induced mouse model, treatment of Vinpocetine reduced the protein levels of cleaved Caspase-1, pro-Caspase-11, cleaved Caspase-11, and IL-1β whereas did not affect the protein levels of pro-Caspase-1 and pro-IL-1β. In the MCD-induced cell model, treatment of Vinpocetine reduced the protein levels of inflammasome components including pro-Caspase-1, pro-Caspase-11, pro-IL-1β in the activated primary hepatocytes and Kupffer cells. These results suggested that Vinpocetine regulated both canonical and non-canonical NLRP3 inflammasome activation pathways.

Conclusion

Treatment of Vinpocetine ameliorated the MCD-induced steatosis symptoms in the NASH mouse model. In addition, treatment of Vinpocetine reduced the inflammation response by the inhibition of inflammation cytokines and inflammasome activation in vivo and in vitro models. More importantly, treatment of Vinpocetine did not show observable toxic effects against the NASH mouse model. These results suggested the therapeutic potential of Vinpocetine for NASH therapy.

Funding

The study was supported by Gastroenterology of key disciplines from Wuxi (ZDXK002); Wuxi Medical Key Talent Project (ZDRC029); Wuxi Sci-Tech Development Fund (CSE31N1603); Wuxi municipal commission of health and family planning project (MS201720); Wuxi municipal commission of health and family planning project (MS201814); Scientific research Surface Project of Wuxi Health Commission (202028); “Two Hundreds” Young and Middle-aged Medical Key Talent Project (HB2020025); “Two Hundreds” Young and Middle-aged Medical Key Talent Project (BJ2020027); “Light of Taihu Lake” Science and Technology Research Project Sponsored by Science and Technology Bureau of Wuxi City (grant nos. Y20212026 and Y20212030).

Conflict of interests

None declared.