He-He-Shu-Yang formula alleviates liver fibrosis by inhibiting hepatic stellate cell activation in vivo and in vitro

Abstract

BACKGROUND

Inhibition of liver fibrosis plays a crucial role in curbing the advancement of chronic disease to cirrhosis and even liver cancer. However, modern medicine currently lacks direct anti-fibrotic drugs. He-He-Shu-Yang formula (HHSY) is a renowned Chinese medicine for the treatment of liver fibrosis. However, its mechanism of action has not been fully unraveled.

AIM

To explore the efficacy and mechanism of action of HHSY through in vitro and in vivo experiments.

METHODS

A liver fibrosis rat model (carbon tetrachloride-induced) was treated with low- or high-dose HHSY (10.42 g/kg or 20.84 g/kg) or with colchicine (1 mg/kg) for 9 weeks. In vitro, LX-2 human hepatic stellate cells (HSCs) were activated using transforming growth factor-β1 and subsequently treated with HHSY-containing serum or a nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) inhibitor. Through high-performance liquid chromatography, histopathology (hematoxylin and eosin, Masson), immunohistochemistry, western blot, and quantitative reverse transcription polymerase chain reaction analyses, we demonstrated that HHSY inhibited HSC activation and suppressed the NOX4/reactive oxygen species (ROS)/nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) pathway.

RESULTS

In vivo, HHSY improved liver function and alleviated liver pathology, including reducing inflammatory cell infiltration, and liver fibrosis in carbon tetrachloride rats. with more significant effects at higher doses. Immunohistochemistry revealed that HHSY could decrease alpha-smooth muscle actin, NOX4, and NLRP3 expression, as well as serum ROS levels (O₂^– and H₂O₂, P < 0.05). Western blot analysis confirmed HHSY also reduced NLRP3 protein levels (P < 0.05). In vitro, HHSY at 1.25% or 2.5% reduced the levels of ACTA2 mRNA, NOX4 protein and NOX4 mRNA, ROS production, and NLRP3 and IL-1β mRNA in activated LX-2 cells (P < 0.05).

CONCLUSION

HHSY effectively treats liver fibrosis, likely by inhibiting HSC activation through the NOX4/ROS/NLRP3 pathway. This underscores HHSY’s clinical relevance as a potential therapeutic option for liver fibrosis.

Key Words: He-He-Shu-Yang formula; Liver fibrosis; Mechanism; Hepatic stellate cells; Nicotinamide adenine dinucleotide phosphate oxidase 4 pathway; Reactive oxygen species pathway

Core Tip: High-performance liquid chromatography identified six key components in He-He-Shu-Yang formula (HHSY) that are effective in reducing liver fibrosis. HHSY has a definite therapeutic effect on liver fibrosis in vivo and in vitro models. HHSY’s mechanism may be through the inhibition of hepatic stellate cell activation via the nicotinamide adenine dinucleotide phosphate oxidase 4/reactive oxygen species/nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 pathways.

INTRODUCTION

Chronic liver disease is a significant global health issue, as it can progress to cirrhosis and hepatocellular carcinoma. In 2021, approximately 1.7 billion patients suffered from chronic liver disease worldwide, with deaths of 1.4 million[1]. Liver fibrosis constitutes an obligate pathological stage in chronic liver disease progression. Preventing liver fibrosis helps decrease the occurrence of cirrhosis and liver cancer, as well as the mortality rate of chronic liver disease. However, liver fibrosis is complex because it involves the accumulation of extracellular matrix (ECM) in the liver and the continuous cross-linking of many components in the ECM, and cross-linked ECM is difficult to degrade[2]. Therefore, current therapeutic options for liver fibrosis are limited, and specific anti-liver fibrosis drugs are currently lacking.

In recent years, numerous studies have validated the efficacy of traditional Chinese medicine (TCM) in managing liver fibrosis[3-5]. Nevertheless, the mechanism of action of TCM in liver fibrosis has not been fully unraveled because of the complexity of TCM preparations. This greatly hinders the development of novel anti-fibrotic TCM drugs. Thus, there is an urgent need to clarify the molecular mechanisms underlying both hepatic fibrogenesis and TCM’s therapeutic mechanisms.

The He-He-Shu-Yang formula (HHSY) has been used clinically for more than 30 years and has demonstrated considerable anti-fibrotic effects in both clinical[6] and experimental studies[7]. It is most commonly prescribed for liver fibrosis or cirrhosis associated with chronic viral hepatitis, non-alcoholic steatohepatitis, and alcoholic liver disease. According to TCM theory, HHSY soothes the liver, strengthens the spleen, promotes blood circulation, and removes blood stasis. Animal studies have also shown that HHSY alleviates liver fibrosis[7]; however, its mechanism remains unclear.

Hepatic stellate cell (HSC) activation plays a crucial role in the progression of liver fibrosis[8]. Chronic liver injury induces HSC activation, leading to excessive ECM production, which in turn promotes the development of liver fibrosis[9-11]. Reduced function of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) group and oxidative stress triggered by reactive oxygen species (ROS) play a crucial role in the progression of hepatic fibrosis. NOX4 facilitates the generation of ROS[12], which in turn can trigger downstream P38 mitogen-activated protein kinase phosphorylation[13], ultimately driving the activation of HSCs in the process of liver fibrosis. Additionally, ROS derived from NOX4 can serve as a second messenger to activate nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes[14], leading to the cleavage of caspase-1 and activation of the pro-inflammatory cytokine interleukin (IL)-1β. Moreover, the secretion of IL-18 exacerbates liver inflammation and damage, thereby activating HSCs to promote the onset and development of liver fibrosis[15].

Based on these findings, we hypothesized that HHSY alleviates liver fibrosis by inhibiting HSC activation through the NOX4/ROS/NLRP3 inflammasome pathway. To test this hypothesis, we established a liver fibrosis rat model using carbon tetrachloride (CCl₄) and an HSC activation model by stimulating LX-2 cells with transforming growth factor-β1 (TGF-β1). Both in vivo and in vitro experiments were conducted to evaluate the effects of HHSY on HSC activation and the associated signaling pathway. These results provide an experimental basis for the clinical application of HHSY.

MATERIALS AND METHODS

Experimental animals

Male Sprague-Dawley (SD) rats, aged 6 weeks and weighing 110-130 g, were obtained from the Animal Centre of Southern Medical University (Guangzhou, Guangdong Province, China; Animal certificate No. 44002100026384). All rats were housed in a controlled environment (22-24 °C, 40%-45% humidity, 12-hour light/dark cycle) and were given ad libitum access to food and water. All experiments involving animals were conducted according to the United States guidelines for the use and welfare of laboratory animals (National Institutes of Health No. 85-23, as amended in 1985) and the ARRIVE 2.0 guidelines, with approval from the Animal Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine, approval No. 2020 064.

Preparation of HHSY extract and quality control

HHSY is comprised of elements, such as Bupleurum chinense DC. (Chai-hu), Aurantii Fructus (Zhi-qiao), Astragalus membranaceus (Huang-qi), Paeonia lactiflora Pall (Bai-shao), and Salvia miltiorrhiza Bunge (Dan-Shen), that were purchased from Kangmei or Zisun Medical Company (Guangzhou, Guangdong Province, China). The HHSY extract was prepared as follows: All herbs were soaked in six times the volume of deionized ultrapure water for 30 minutes and then decocted for 1 hour two times. The extraction solutions were combined and filtered into a 2-L beaker. The filtrated extract was concentrated to 2.084 g/mL using an R2005KB rotary evaporator (Senco, Shanghai, China) at approximately 65 °C, aliquoted, and stored at -20 °C. For quality control of HHSY, high-performance liquid chromatography (HPLC) fingerprint analysis was performed for specific ingredients. An Agilent Eclipse XDB-C18 chromatographic column (5 μm, 4.6 mm × 150 mm; Agilent Technologies China, Beijing, China) was used, with the following elution settings: Mobile phase acetonitrile (A) -0.1% phosphoric acid (B) and gradient elution (0-5 minutes, 5% A; 5-10 minutes, 5%-20% A; 10-40 minutes, 20%-25% A; 40-50 minutes, 25%-100% A; 50-60 minutes, 50%-100% A; 60-65 minutes, 5%-50% A), flow rate: 1 mmol/L/minute. Detection was performed at a wavelength of 230 nm, with the column held at room temperature (25 ± 2 °C) and an injection volume of 10 μL.

Cell culture and treatments

LX-2 human HSCs (BNCC341818; BeiNa Biological Company) were seeded at a density of 3.6 × 10⁴ cells/cm² in 6-well plates. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM, C11995500BT; Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10% fetal bovine serum, in a 5% CO₂ incubator at 37 °C. Before the experiment, LX-2 cells were starved in DMEM without fetal bovine serum for 12 hours. To induce HSC activation, LX-2 cells were stimulated with 5 ng/mL TGF-β1 for 24 hours. Cells were divided into seven groups: Control (fresh DMEM), model (5 ng/mL TGF-β1), HHSY serum (5 ng/mL TGF-β1 + serum containing 0.125%, 0.5%, 1.25%, 2.5%, or 5% HHSY), and NOX4 inhibitor (5 ng/mL TGFβ1 + 10 nM GKT137831), with each treatment administered for 24 hours. All experiments were performed with three biological replicates and three technical replicates per biological replicate, resulting in a total sample size of 9 per group.

Animal model establishment and treatments

Based on preliminary experimental results, the hepatic fibrosis model was established by intraperitoneally injecting SD rats with 30% CCl₄ in olive oil (3:7 v/v; 10 mL/kg body weight) every 3 days for 9 weeks. Fifty SD rats were randomly divided into the following five groups (n = 10 rats/group): Control, model, Low-dose HHSY (HHSY-L), High-dose HHSY (HHSY-H), and colchicine. Rats in the control group were intraperitoneally injected with olive oil (1 mL/kg) every 3 days for 9 weeks, whereas those in the other groups were injected with 30% CCl₄ in olive oil as mentioned above. The day after injection, rats in the control and model groups were administered 0.1 mL/kg distilled water via orogastric gavage once daily for 9 weeks, whereas those in the HHSY-L and -H groups were given 10.42 g/kg and 20.84 g/kg HHSY, respectively. Rats in the colchicine group were given 1 mg/kg colchicine via oral gavage once daily for 9 weeks.

Liver index and histopathological examination

After the rats were euthanized by intraperitoneal injection of 10% chloral hydrate (freshly prepared) at a dose of 3.5 mL/kg, their livers were collected, and the liver index was calculated by dividing the wet weight of the liver by the body weight, multiplied by 100%. Serum was prepared from rat abdominal aorta blood via centrifugation at 3000 rpm (15 minutes, 4 °C). Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were measured via an automatic biochemical analyzer (cobas 6000, Roche Diagnostics, Basel, Switzerland). Liver tissues were cut into 15’ 15’ 5-mm blocks, which were fixed with 10% formalin, processed using routine histopathological techniques, and sliced into 4-mm sections. The sections were deparaffinized with xylene and rehydrated with 100% ethanol for 5 min, followed by 80% ethanol for another 5 minutes. Then, the sections were stained with hematoxylin and eosin staining kit (Beyotime Institute of Biotechnology, Shanghai, China) and Masson trichrome stain (Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China) according to the manufacturer’s instructions. The sections were imaged using an optical microscope at a magnification of 200’ and assessed in a blinded manner by two independent third-party pathologists who were unaware of the treatment assignments.

Immunohistochemistry

Sections were deparaffinized and rehydrated using standard protocols, permeabilized in 0.4% Triton X-100 and 1% bovine serum albumin for 2 hours, and incubated with the following primary antibodies at 4 °C for 72 hours: Anti-α-smooth muscle actin (α-SMA) antibody (ab7817,Abcam, Cambridge, MA, United States; diluted 1:10000 in 1% bovine serum albumin dissolved in phosphate-buffered saline, NOX4 antibody (ab133303, Abcam; diluted 1:2000), NLRP3 antibody (ab263899, Abcam; diluted 1:500). Following the wash step, the slices were exposed to secondary antibodies at a temperature of approximately 25 ± 2 °C for a duration of 2 hours. The secondary antibody used was a highly sensitive horseradish peroxidase-conjugated anti-mouse/rabbit IgG polymer (Zhongshan Golden Bridge Biotechnology, dilution 1:500). Subsequently, the segments underwent a washing process, were dried, and were positioned onto slides. The sections were observed at × 200 magnification using a Leica confocal microscope and stained cells in a random microscopic field were counted. α-SMA expression was semi-quantitatively analyzed by measuring the average optical density (AOD) using the ImageJ software (version 1.53, NIH, Bethesda, MD, United States). Antibody against Col1A1 (1:200; sc2597; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States) was used for immunohistochemistry (IHC) via the Envision two-step procedure in SD rats with CCl₄-induced liver fibrosis. Cytoplasmic deposition of granules with a brownish-yellow hue indicated Col1A1 expression.

Immunofluorescence staining

LX-2 cells grown on coverslips were induced with TGF-β1 (5 ng/mL) for 24 hours, fixed in 4% paraformaldehyde, and incubated with anti-α-SMA antibody (diluted 1:200, 14395-1-AP; Proteintech Group, Wuhan, Hubei Province, China) at 4 °C overnight. After a wash with phosphate-buffered saline, the cells were incubated with fluorescein isothiocyanate-conjugated fluorescent secondary antibodies [goat anti-rabbit IgG (H + L) FITC conjugate, Zhongshan Golden Bridge Biotechnology, dilution 1:500] in the dark for 2 hours. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (ab104139; Abcam). Finally, the TGF-β1-induced activated LX-2 cells were observed and imaged using a fluorescence microscope (Olympus, Tokyo, Japan).

Western blotting

Liver tissues were homogenized in a 1% phenylmethylsulfonyl fluoride (Sigma-Aldrich, Solarbio)-containing radioimmunoprecipitation assay buffer (Beyotime, Thermo Fisher Scientific) with phosphatase inhibitors (Roche; Cell Signaling Technology, Beverly, MA, United States). Proteins (20 μg per lane) were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Merck, IPVH00010). The membranes were blocked with 5% bovine serum albumin, incubated with primary antibodies (1:1000; Cell Signaling Technology) at 4 °C overnight including anti-NOX4 (ab133303,Abcam; diluted 1:2000), anti-NLRP3 (ab263899, Abcam; diluted 1:500) and anti-Caspase1 (sc-56036, Santa Cruz Biotechnology; diluted 1:1000). After washing with Tris- buffered saline, the membranes were incubated with appropriate secondary antibodies at room temperature for 2 hours. Blots were developed with Clarity ECL (Bio-Rad, Hercules, CA, United States) and imaged using ChemiDoc Touch Imaging System. Band intensities were analyzed using ImageLab software (v6.1, Bio-Rad Laboratories), with β-actin as an internal control.

Quantitative reverse transcription polymerase chain reaction

Total RNA was isolated from various types of cells and tissues using TRIzol Reagent and quantified using a Nanodrop 2000 spectrophotometer. cDNA was synthesized using a reverse transcription kit (lot No. RR360A; Takara Bio, Shiga, Japan). Quantitative polymerase chain reaction (PCR) was performed using a SYBR Green kit (RR820A; Takara Bio) in an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific). The thermal cycling program was as follows: Initial denaturation at 95 °C for 15 minutes, followed by 40 cycles at 95 °C for 15 seconds, 60 °C for 60 seconds, and 72 °C for 10 minutes. Relative target gene expression was quantified using the 2^-ΔΔt method, using the housekeeping gene GAPDH as a reference gene. Primers were designed using ABI Primer Express 3.0 and synthesized at Sangon Biotech, Shanghai, China (Table 1).

Table 1 Quantitative reverse transcription polymerase chain reaction primer sequences.

Target gene	Primer	Sequence (5’-3’)
NLRP3	Forward primer	5’-GCTTGCCGACGATGCCTTCC-3’
	Reverse primer	5’-GCTGTCATTGTCCTGGTGTCTTCC-3’
ACTA2	Forward primer	5’-TCGTGCTGGACTCTGGAGATGG-3’
	Reverse primer	5’-CCACGCTCAGTCAGGATCTTCATG-3’
NOX4	Forward primer	5’-TCACAGCCTCTACATATGCAAT-3’
	Reverse primer	5’-CAGCAGCATGTAGAAGACAAAG-3’
CASP1	Forward primer	5’-GAAGAAACACTCTGAGCAAGTC-3’
	Reverse primer	5’-GATGATGATCACCTTCGGTTTG-3’
IL-1β	Forward primer	5’-GCCAGTGAAATGATGGCTTATT-3’
	Reverse primer	5’-AGGAGCACTTCATCTGTTTAGG-3’
GAPDH	Forward primer	5’-GAAGGTGAAGGTCGGAGTC-3’
	Reverse primer	5’-GAAGATGGTGATGGGATTTC-3’

Cell viability assay

To determine the non-cytotoxic HHSY serum concentration, cell viability was assessed using a Cell Counting Kit-8 (CCK-8, C0038, Beyotime Biotechnology, Shanghai, China). In brief, LX-2 cells were inoculated in a 96-well plate at 5000 cells/well and cultured for 24 hours. After treatment with HHSY at concentrations of 0.125%, 0.5%, 1.25%, 2.5%, and 5% for 24 hours, 10 μL CCK-8 solution was added to each well and the plate was incubated in a constant temperature incubator at 37 °C for 2 hours. The absorbance value at 450 nm was measured using a microplate reader. All samples were analyzed at least three times. The optical density (OD) values were measured and cell viability was calculated as follows: Cell viability = (detection well OD value - blank well OD value)/(control well OD value - blank well OD value).

Statistical analysis

GraphPad Prism 8 software GraphPad Software, San Diego, CA, United States) was used for data graphing and statistical analysis. Normal distribution was assessed utilizing the Shapiro-Wilk test and normally distributed data are reported as mean ± SD. Means of two groups with a normal distribution and homogeneous variance were analyzed using a two-independent-sample t-test. Analysis of variance was used for multiple groups, accompanied by Tukey’s test for honestly significant differences to evaluate pairwise comparisons. For datasets that do not follow a normal distribution pattern, the results were presented with the median and interquartile range. To assess statistical differences among various groups, the Kruskal-Wallis test was employed, succeeded by Dunn’s post-hoc analysis for examining pairwise contrasts. Statistical significance was set to P < 0.05.

RESULTS

Quality control of HHSY using HPLC fingerprinting

To characterize the HHSY extract, six compounds were identified using HPLC analysis. The main components of the HHSY extract according to the main peaks in the HPLC-ultraviolet chromatogram were identified as paeoniflorin, isoflavone glucoside, glycyrrhizin, naringin, hesperidin, and neohesperidin (Figure 1).

Figure 1 High-Performance liquid chromatography chromatograms of He-He-Shu-Yang formula extracts. Peak 1: Paeoniflorin; Peaks 2: Isoflavone glucoside; Peak 3: Glycyrrhizin; Peak 4: Naringin; Peak 5: Hesperidin; Peak 6: Neohesperidin.

HHSY alleviates CCl₄-induced liver fibrosis in SD rats

After a 9-week intervention period, the mortality rate among rats was relatively low. Only one death was recorded in each of the model, HHSY-H, and colchicine groups, whereas no deaths were observed in the other groups. Rats in the control group exhibited liveliness, activeness, and prompt reactions. Conversely, rats in the model group displayed decreased activity levels and a preference for remaining motionless and congregating in groups. However, rats in the HHSY-L, HHSY-H, and colchicine groups demonstrated improved activity and responsiveness.

As shown in Figure 2A and B, rats in the model group exhibited a substantial decline in body weight and a marked increase in the liver index. Treatments with HHSY-H and colchicine resulted in greater improvements in body weight compared to the model group, although the differences were not statistically significant. However, compared with the model group, both treatment groups showed a significant reduction in the liver index (Figure 2B). CCl₄ model rats exhibited significantly greater increases in serum ALT and AST levels compared to those in the control group (Figure 2C and D). The HHSY-L, HHSY-H, and colchicine treatments significantly decreased the serum ALT and AST levels, with no statistically significant distinction between the HHSY-H and colchicine groups. Histological changes are shown in Figure 2E. Compared to those in the control group, liver tissues from the model group exhibited substantial inflammatory cell infiltration and extensive fibrous tissue proliferation in the portal area and around the central vein. Furthermore, the liver lobule structure was compromised, resulting in the disappearance of liver cords and the formation of pseudolobules. Conversely, fibrotic damage was markedly improved in the treatment groups, with thinner fibrous septa and reduced inflammatory infiltration. Rats in the HHSY-H and colchicine groups exhibited minimal inflammatory cell infiltration and a greater degree of improvement than those in the HHSY-L group. Therefore, only the HHSY-H treatment was selected for subsequent mechanistic experiments.

Figure 2 He-He-Shu-Yang formula alleviates carbon tetrachloride-induced liver fibrosis in Sprague-Dawley rats. ^aP < 0.05, ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. Data are presented as median (inter quartile range), n = 9-10 rats/group. Rats were divided into five treatment groups: Control, model, low-dose He-He-Shu-Yang formula, high-dose He-He-Shu-Yang formula, and colchicine. A: Body weight in each group; B: Liver index in each group; C: Serum alanine aminotransferase level in each group; D: Serum aspartate aminotransferase level in each group; E: Representative images of liver pathology. Hematoxylin and eosin and Masson staining of liver tissues (magnification: 100’, scale bar: 100 μm). HHSY-L: Low-dose He-He-Shu-Yang formula; HHSY-H: High-dose He-He-Shu-Yang formula; CCl₄: Carbon tetrachloride; HE: Hematoxylin and eosin.

HHSY inhibits HSC activation in vivo

HHSY treatment effectively suppressed the expression of α-SMA, a marker of HSC activation in the rat liver. IHC staining revealed a limited presence of α-SMA in the liver of rats in the control group (Figure 3A). Conversely, rats in the model group exhibited heightened α-SMA expression in the hepatic sinusoid, portal vein, and portal area. Remarkably, animals in the HHSY and colchicine groups displayed a substantial reduction in α-SMA expression in these areas. The AOD of α-SMA showed a significantly greater increase in the model group compared to the control group (P < 0.001). Compared to that in the model group, both the HHSY and colchicine groups exhibited a significantly greater reduction in α-SMA AOD (P < 0.001; Figure 3B); however, the difference between the two groups was insignificant (P = 0.977).

Figure 3 He-He-Shu-Yang formula inhibits hepatic stellate cell activation in rats with carbon tetrachloride-induced fibrosis. ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. A: Representative images of α-smooth muscle actin immunohistochemical staining in the indicated treatment groups (bar = 50 μm); B: Average optical density of α-smooth muscle actin in each group. HSC: Hepatic stellate cell; α-SMA: Α-smooth muscle actin; HHSY: He-He-Shu-Yang formula; AOD: Average optical density; A.U.: Arbitrary unit.

HHSY blocks NLRP3 inflammasome activation in liver fibrotic rats

IHC staining results showed that NOX4 expression (brown area in Figure 4A) was significantly increased by CCl₄ treatment, and this increase was significantly suppressed by HHSY and colchicine (Figure 4A). ROS encompass various compounds, including superoxide anions (O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH). As shown in Figure 4B-D, serum levels of O₂^– and H₂O₂ were strongly increased in the CCl₄ model group, whereas the OH clearance rate was decreased. HHSY strongly reduced the serum levels of O₂^- and H₂O₂ and enhanced the OH clearance rate. No significant differences were found between the HHSY and colchicine groups. IHC revealed that NLRP3 expression was strongly enhanced in rats in the model group but significantly suppressed in animals in the HHSY and colchicine groups (Figure 4E). Western blot results showed that the protein levels of NLRP3 and caspase-1, which are related to the NLRP3 inflammasome pathway, were significantly increased in the model group. Conversely, the decrease in the expression of these two proteins was significantly more in both the HHSY and colchicine groups than in the model group (Figure 4F).

Figure 4 He-He-Shu-Yang formula inhibits the nicotinamide adenine dinucleotide phosphate oxidase 4/reactive oxygen species/nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome pathway in rats with carbon tetrachloride-induced fibrosis. ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. A: Representative images of nicotinamide adenine dinucleotide phosphate oxidase 4 immunohistochemistry staining in each group (bar = 100 μm); the brown area shows nicotinamide adenine dinucleotide phosphate oxidase 4 expression; B: Serum level of superoxide anion (O₂^-) in each group; C: Serum level of hydrogen peroxide (H₂O₂) in each group; D: Hydroxyl radical (OH) clearance rate in each group; E: Representative images of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 immunohistochemistry staining (bar = 100 μm); F: Protein levels of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 and cleaved caspase-1 in the different groups, as determined using western blotting. HHSY: He-He-Shu-Yang formula; NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4; NLRP3: Nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3.

HHSY inhibits TGF-β1-induced LX-2 activation in vitro

CCK-8 results showed that, compared with that in the control group, the viability of LX-2 cells exposed to serum containing 0.125%, 0.5%, 1.25%, or 2.5% HHSY was above 90%, with no significant differences among the treatment groups. However, the cell viability of cells treated with serum containing 5% HHSY was significantly reduced. Therefore, in subsequent experiments, HHSY was used at 0.125%, 0.5%, 1.25%, and 2.5%. The alterations were notably more in the cells of the model than in those of the control group, accompanied by a substantial increase in cell proliferation as indicated by the increase in the average fluorescence intensity of α-SMA in immunofluorescence analysis (Figure 5A and B). Treatment with HHSY at 1.25% or 2.5% and NOX4 inhibition resulted in a significantly lower average fluorescence intensity of α-SMA compared to the level in the model group (P < 0.05). ACTA2 mRNA levels assessed using quantitative reverse transcription PCR showed similar alterations as those observed in the immunofluorescence experiments (Figure 5C).

Figure 5 He-He-Shu-Yang formula inhibits transforming growth factor-beta 1-induced activation of the human hepatic stellate cell line (LX2). ^bP < 0.01 vs control group; ^dP < 0.01 vs model group. Data are mean ± SD. A: Representative images of immunofluorescence staining of α-smooth muscle actin (α-SMA) in LX2 cells; B: Mean fluorescence intensity of α-SMA; C: Relative ACTA2 (which encodes α-SMA) mRNA levels. α-SMA: Α-smooth muscle actin; AU: Arbitrary unit.

Mechanistic validation: NOX4/ROS/NLRP3 pathway

Considering the potential influence of excess NOX4 on HSC activation, we measured NOX4 Levels in TGF-β1-induced LX-2 cells. As anticipated, NOX4 protein and NOX4 mRNA levels increased significantly in response to TGF-β1 stimulation (Figure 6A-C). Importantly, NOX4 accumulation in TGF-β1-induced LX-2 cells was markedly diminished by treatment with HHSY at 1.25% or 2.5% and by NOX4 inhibition. Notably, there was no discernible difference between the 2.5% HHSY group and the NOX4 inhibitor group. The mean fluorescence intensity of ROS in TGF-β1-induced LX-2 cells was notably higher than that in the control group (P < 0.01; Figure 6D and E) but was significantly decreased by 1.25% and 2.5% HHSY and NOX4 inhibition (P < 0.01). Additionally, HHSY and NOX4 inhibition suppressed NLRP3 and IL-1β mRNA levels in LX2 cells induced by TGF-β1 (Figure 6F and G).

Figure 6 He-He-Shu-Yang formula inhibits nicotinamide adenine dinucleotide phosphate oxidase 4 expression, intracellular reactive oxygen species generation, and nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome activation in transforming growth factor-beta 1-induced LX2 cells. ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs model group. A: Representative western blot of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression in each group. Protein levels were normalized to β-actin; B: Quantitation of NOX4 expression in each group based on the blot in panel A; C: Relative mRNA levels of NOX4 in the various groups; D: Intracellular reactive oxygen species levels as assessed using fluorescence microscopy; E: Mean fluorescence intensity of reactive oxygen species; F: Relative mRNA levels of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 in each group; G: Relative mRNA levels of Interleukin-1β in each group. NOX4: Nicotinamide adenine dinucleotide phosphate oxidase 4; TGF-β: Transforming growth factor-beta 1; ROS: Reactive oxygen species; NLRP3: Nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3; IL-1β: Interleukin-1β.

DISCUSSION

In this study, we evaluated the efficacy of HHSY in alleviating liver fibrosis in vivo and in vitro. Our results revealed that HHSY acts by inhibiting HSC activation via the NOX4/ROS/NLRP3 inflammasome pathway. In what follows, we discuss our main findings. First, HPLC for quality control identified six key components in HHSY: Isoflavone glucoside, paeoniflorin, glycyrrhizin, naringin, hesperidin, and neohesperidin. Studies have shown that these six components[16-20] can ameliorate liver injury and fibrosis. Among its components, paeoniflorin[21] has been reported to inhibit HSC activation and reduce ECM deposition both in vivo and in vitro, providing a theoretical basis for HHSY’s anti-fibrotic effects. Consistent with these component-specific findings, our data confirmed that HHSY alleviated liver fibrosis by inhibiting HSC activation. Activated HSCs can transform into myofibroblasts that secrete matrix proteins, thereby driving liver fibrogenesis[11]. Increasing evidence indicates that oxidative stress and inflammatory cytokines can activate HSCs[22], which then produce ECM and promote liver fibrosis. In our CCl₄ rat model - where CCl₄ is metabolized to the CCl₃ free radical that damages hepatocytes, stimulates inflammatory processes, and activates HSCs - HHSY significantly decreased the liver index, serum ALT and AST levels, inflammatory cell infiltration, and fibrotic tissue proliferation in the liver. These results suggest that HHSY alleviates liver injury and fibrosis. More importantly, HHSY was found to effectively suppress the expression of α-SMA, a marker of HSC activation. In CCl₄-induced fibrotic rats, HHSY substantially reduced the α-SMA-positive area in the liver. In vitro, HHSY inhibited TGF-β1-induced LX2 cell activation. Treatment with 1.25% and 2.5% HHSY significantly decreased α-SMA expression as indicated by fluorescence and quantitative reverse transcription PCR analyses. Crucially, the findings demonstrated no significant statistical difference between the HHSY group and the colchicine control group, further supporting HHSY’s anti-fibrotic potency.

Second, we found that HHSY effectively reduces liver fibrosis by significantly mediating the NOX4/ROS/NLRP3 pathway, revealing a key molecular mechanism of action of HHSY. Liver inflammation and oxidative stress both contribute to HSC activation and liver fibrosis[23]. NOX4, as a main source of ROS, can cause the excessive accumulation of oxidation products, which consequently promotes HSC activation and contributes to liver fibrosis[24]. Furthermore, NLRP3 inflammasome activation can trigger caspase-1 activation and the release of mature IL-1β and IL-18, resulting in an inflammatory cascade that further activates HSCs and promotes liver fibrosis[25]. Recent research has demonstrated that ROS-mediated oxidative stress can activate the NLRP3 inflammasome pathway, resulting in HSC proliferation and activation[26]. This evidence shows that NOX4/ROS and NLRP3 can crosstalk to activate HSCs. In this study, HHSY treatment decreased NOX4 and NLRP3 expression as indicated by IHC staining results, as well as serum O₂^– and H₂O₂ levels in CCl₄-induced liver fibrotic rats. In TGF-β1-induced LX-2 cells, HHSY treatment not only decreased the mRNA levels of NOX4, NLRP3, and IL-1β, but also showed a significantly decreased ROS production. Importantly, there were no major variations observed between the HHSY group and those treated with NOX4 inhibitors. These results suggest that HHSY alleviates liver fibrosis in vivo and in vitro by inhibiting HSC activation via the NOX4/ROS/NLRP3 pathway.

Notably, the promising anti-fibrotic effects of HHSY demonstrated in this study are supported by an ongoing nationwide, multi-center randomized controlled trial in patients with chronic hepatitis B-related liver fibrosis. The translational potential of HHSY is underscored by the clinically achievable doses used in this preclinical model and its multi-target mechanism. Overall, with forthcoming clinical evidence, these findings position HHSY as a strong complementary therapy candidate for chronic hepatitis B-related liver fibrosis.

The research encountered several constraints. In the in vitro experiments, only one cell line, LX-2, was used, and primary HSCs were not used for validation. In future, we plan to carry out experiments with other relevant cells for further verification. In addition, because of time and funding constraints, we did not use NOX4 overexpression or knockout cells to observe the effect of HHSY on downstream NLRP3 inflammasome signaling pathways. In the future, these types of experiments ought to be carried out.

CONCLUSION

Overall, our findings demonstrate that HHSY improves liver fibrosis in vitro and in vivo by inhibiting HSC activation through the NOX4/ROS/NLRP3 pathway. Importantly, this study provides a novel therapeutic strategy for liver fibrosis - an area with limited pharmacologic options - by demonstrating the potential of a multi-target traditional medicine to ameliorate both oxidative stress and inflammatory activation, key drivers of fibrosis. This finding may pave the way for the use of HHSY as an antifibrotic therapy.