Antiliver Fibrosis Formula of Fuzheng Huayu Alleviates Inflammatory Response

Fuzheng Huayu's (FZHY) formula ameliorated liver fibrosis in clinical and experimental practice. Based on the close link between fibrosis and inflammation, its anti-inflammatory effect and related mechanisms were explored in this present study. With the aid of the inflammatory macrophage model, FZHY significantly blocked nitrite accumulation without observable cytotoxicity due to its suppression of inducible nitric oxide synthase (iNOS) gene and protein expressions in a concentration-depended manner. Proinflammatory mediators including IL-6, CD86, and CD40 were also restrained by FZHY. Interestingly, FZHY induced anti-inflammatory mediators heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions simultaneously. Downregulation of iNOS and miR-155 and upregulation of PPAR-γ were also observed in CCl4-induced liver fibrosis mice upon FZHY administration. Mechanically, FZHY strikingly eliminated the phosphorylation of STAT1 and MAPK. Taken together, FZYH regulated the balance of proinflammatory and anti-inflammatory mediators partially via modulating STAT1/MAPK pathways and the miR-155/PPAR-γ axis.


Introduction
Hepatic fbrosis is a chronic wound-healing response characterized by infammation [1]. Genetic changes, hepatitis virus infections, excessive alcohol consumption, lipid metabolic disorders, and autoimmune diseases trigger continuous liver injury and subsequent chronic infammation that lead to liver fbrosis [2,3]. Understanding the underlined infammatory mechanisms is critical to developing strategies to control fbrosis [4].
Hepatic macrophages are the primary immune cells, consisting of liver-resident macrophages, monocytederived macrophages, and Kupfer cells that trigger liver infammatory response and also play a crucial role in the pathologic progress of fbrosis [5]. Traditionally, macrophages can be polarized into proinfammatory/M1 macrophages or anti-infammatory/M2 macrophages upon diferent microenvironmental stimulations. M1 macrophages predominantly express inducible nitric oxide synthase (iNOS) and secrete classical proinfammatory cytokines such as interleukin (IL)-6 and IL-1β to exaggerate the infammatory response induced by T helper (T)-1 signals such as lipopolysaccharide (LPS) and interferon (IFN)-c. Accumulating evidence strongly implies that iNOS-derived nitric oxide (NO) has been associated with the pathogenesis of liver diseases during infammatory conditions. Tese infammatory mediators also emerge as vital profbrotic hubs. IL-1β promotes liver fbrosis partially in an IL-17-dependent manner [6]. Tus, targeting macrophages and inhibition of M1 macrophage-led infammation development are approaches to interfere with fbrosis [7].
MicroRNAs (miRNAs) are small, noncoding RNAs that negatively control target gene expressions by promoting degradation or translational inhibition. miR-155 exerts a proinfammatory efect during the progress of hepatic fbrosis in immune cells [8]. PPAR-c is involved in the progression of liver fbrosis as one of the target genes of miR-155 [9]. CCl 4 administration decreases the expression of PPAR-c in liver tissue and the antifbrotic efect of crocin partly via enhancing PPAR-c to mediate infammatory response and fbrogenic events [10].
Tus, in this present study, we evaluated the antiinfammatory efect and relevant mechanisms of FZHY.

High-Performance Liquid Chromatography (HPLC)
Assay. FZHY standardization was performed using HPLC fngerprinting with chemical standard compounds such as sodium danshensu, salvianolic acid B (two compounds isolated from Radix Salvia Miltiorrhizae), and adenosine (a compound isolated from Cordyceps) according to Chinese Pharmacopoeia (2015 edition).

Cell
Culture. RAW 264.7 cells from the American Type Culture Collection (ATTC, Rockville, MD) were cultured in RPMI 1640 medium supplemented with 10% FBS. Te cell passages below 10 were used, and three replicates were performed in each experiment.

Cell Viability.
Te efect of FZHY on the proliferation of RAW 264.7 cells was detected by the MTT assay. RAW 264.7 cells were incubated into 96-well plates at a density of 10000 cells/well and were allowed to adhere overnight. Cells were treated with FZHY in diferent concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1000 μg/mL) and under 1400W (50 μM), respectively. After 24 hours, 0.5 mg/ml MTT was added to each group and was incubated for additional 4 hours. Te supernatant of each group was discarded and then the absorbance at 490 nm with formazan-DSMO dissolution was read. Te cell availability of each group was calculated compared to the cell availability of the control group as 100%.

Nitrite
Assay. RAW 264.7 cells were seeded into 96-well plates at a density of 100,000 cells/well and were divided into the control group, the model group, FZHY treatment groups (25, 50, 100, and 200 μg/mL), and the positive drug group 1400W (50 μM, a selective inhibitor of iNOS), respectively. After serum-free treatment for 24 h, the control group was treated with serum-free 1640 medium, and the model group was stimulated with LPS (100 ng/mL)/IFN-c (100 U/mL), while the FZHY administration groups were treated with 25, 50, 100, and 200 μg/mL and stimulators LPS (100 ng/mL)/ IFN-c (100 U/mL). After 24 hours, 100 μL of the supernatant of each group was collected and added to 100 μL of Griess reaction reagent for 10 mins and then was read at 540 nm using a microplate reader. Ten, the nitrite content in the cell supernatant was calculated using the nitrate standard curve.

Animal Experiment.
Te murine liver fbrosis model induced by CCl 4 and administration with or without FZHY was proceeded in a previous study [20], and preserved livers were used for further experiments to detect the infammation-relevant mediators.
2.7. qPCR Analysis. RAW 264.7 cells were cultured and divided into the control, model, and FZHY treatment groups (100 and 200 μg/mL), respectively. After being allowed to adhere overnight, cells were treated with serum-free 1640 medium for 24 h. Te samples of cells and liver tissues were collected, and the total RNA was extracted by the Trizol method, and miR-155, iNOS, CD86, CD40, IL-6, PPAR-c, and HO-1 were detected. Te primers of qRT-PCR for iNOS, CD86, CD40, IL-6, PPAR-c, and HO-1 are listed in Table 1.

Western Blot
Analysis. RAW 264.7 cells were cultured in 30 mm culture dishes with 1 × 10 6 cells. Te protein samples of each group were collected, and western blotting was performed after protein denaturation. Te protein expressions of iNOS, HO-1, CD40, CD86, and IL-6 compared with β-actin and the phosphorylation levels of p38, JNK, ERK, and STAT1 compared with total p38, JNK, ERK, and STAT1 were determined.

Statistical
Evaluation. Data were presented as the mean ± SD of results obtained from at least three experiments. Data were assessed by ANOVA analysis and the ttest. P < 0.05 was considered as statistically signifcant.
iNOS has been used as the major biomarker for the defnition of M1 proinfammatory macrophages, so we further examined the efect of FZHY at dosages of 25, 50, 100, and 200 μg/mL on nitrite accumulation, the stable oxidative metabolite of nitric oxide, and in the supernatant from diferently treated cells. FZHY inhibited nitrite accumulation induced by LPS plus IFN-c in a concentrationdependent manner (Figure 1(b)). As expected, FZHY inhibited iNOS expressions at gene and protein levels in a concentration-dependent manner on infammatory macrophages (Figures 1(c) and 1(d)). 1400W, a well-known inducible nitric oxide synthase (iNOS) selective inhibitor, reduced nitrite production by 97.45% at 50 μM compared to the model group induced by LPS plus IFN-c.
Compared with the normal group, the infltration of infammatory cells and collagen deposition in the portal area were signifcantly increased in CCl 4 -induced liver fbrosis mice, while FZHY presented the prevention and curing efects on CCl4-induced liver infammation and fbrosis [20]. iNOS also altered its expression in liver fbrosis. iNOS defciency improved liver infammation and genes encoding collagen, leading to decrease fbrosis [22]. We continued to test iNOS in the liver tissues from CCl 4 -induced liver fbrosis mice with and without FZHY administration. Results demonstrated that FZHY administration did reduce iNOS expression in liver tissues (Figures 1(e) and 1(f )).
Based on these results, FZHY strongly attenuated iNOS in infammatory macrophages and liver tissues from CCl 4induced liver fbrosis mice.

FZHY Enhanced Expression of Anti-Infammatory Enzyme HO-1.
HO-1 is the inducible and rate-limiting enzyme in heme catabolism and exhibits anti-infammatory functions to resolve cellular oxidative stress and infammatory cascade reaction. LPS failed to induce iNOS production in HO-1-overexpressing cells suggesting that HO-1 protected RAW 264.7 cells from infammation damage [23]. Terefore, we examined the efect of FZHY on HO-1 using qPCR and western blot analysis on macrophages. Results showed that FZHY strikingly increased expressions of HO-1 at gene and protein levels (Figure 2).
Tus, we next investigated whether FZHY inhibited iNOS expression by regulating STAT1/MAPK pathways. LPS plus IFN-c increased the phosphorylation levels of MAPK and STAT-1. FZHY treatment concentration dependently abrogated phosphorylation of STAT1 and MAPK ( Figure 4).
Tese data suggested that STAT1/MAPK pathways were involved in the FZHY-led efect.

3.5.
FZHY Modulated miR-155/PPAR-c Axis. miR-155/PPAR-c axis regulated the progress of infammation and liver fbrosis [9]. Under the infammation condition, the level of miR-155 was notably boosted. However, FZHY dramatically struck the elevated level of miR-155 and upregulated expression of its target gene PPAR-c on macrophages and liver tissues from CCl 4 -induced liver fbrosis mice ( Figure 5).

Chemical Quality Control of FZHY by HPLC Analysis.
We identifed three compounds as the chemical quality control of FZHY by HPLC analysis (Figure 6). Te content of sodium danshensu (8.3%), salvianolic acid B (13.25%), and adenosine (3.95%) in FZHY met the requirements of Chinese Pharmacopoeia.

Discussion
Infammation is a key component and a contributor to profbrogenic progress. Increasing evidence showed that anti-infammatory therapy exerted its efect in the treatment of liver fbrosis [26]. Targeting chronic infammation in the context of fbrogenesis might lead to potential antifbrotic therapies. Macrophages play a central role in the progression of liver infammation and fbrosis progression [27]. M1 macrophages induced enzymes and secreted cytokines to regulate fbrogenesis [28]. Tus, controlling M1 macrophage polarization during fbrosis provides a crucial strategy.
LPS plus IFN-c can activate M1 macrophages that exert a proinfammatory phenotype. iNOS is a signifcant marker of M1 macrophages. Excessive NO, a gas signal molecule with high reactive properties, produced by iNOS results in oxidative and nitroxidative stress under infammatory conditions. Tese fndings demonstrated that FZHY suppressed the expression of iNOS at gene and protein levels in a concentration-dependent manner. Te accumulation of nitrite and the steady production of NO are also reduced by FZHY (Figure 1). Tus, FZHY's anti-infammatory activity depends on its inhibition of NO and iNOS. Other M1 markers, including CD86, CD40, and IL-6, were also diminished upon FZHY administration (Figure 3).
A network pharmacology approach and a cell-based assay revealed that schisandrin B, salvianolic acid A, and  Evidence-Based Complementary and Alternative Medicine kaempferol from FZHY could bind to PPAR-c [19]. We found that proinfammatory miR-155 increased while its target gene PPAR-c decreased upon stimulation by LPS in IFN-c or CCl 4 -induced liver fbrosis mice, while FZHY reduced the level of miR-155 and upregulated the expression of PPAR-c ( Figure 5).

Conclusions
Collectively, our fndings demonstrated that FZHY exerted an anti-infammatory efect on LPS plus IFN-c -induced infammation with modulation of proinfammatory and anti-infammatory mediators via MAPK/STAT-1 signaling pathways and miR-155/PPAR-c axis (Figure 7). Future studies are needed to elucidate active compounds from FZHY and core targets and pathways in depth underlying the relationship between infammation and fbrosis.

Data Availability
Te data used to support the fndings of this study are included in the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.