Qinggan Huoxue Recipe Protects against Experimental Alcoholic Liver Fibrosis through CXCL16 Inhibition

Background Qinggan Huoxue recipe (QGHXR), a traditional Chinese medicinal formula, has a protective effect against liver fibrosis. However, the underlying mechanisms remain unclear. Objective This study investigated the antifibrotic role of QGHXR and its underlying mechanisms. Methods The composition of QGHXR was determined using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Female C57BL/6J mice were fed either a Lieber–DeCarli liquid diet or pair-fed control diet and intraperitoneally injected with CCl4 for 8 weeks (n = 8). In week 5, the mice were administered 100, 200, and 400 mg/kg QGHXR via oral gavage daily for 4 weeks. Results UPLC-MS result showed that QGHXR contained 45 compounds including salvianolic acid A, scutellarin, baicalin, rutin, and chai saponin D. QGHXR alleviated pathological alterations in the liver. The alanine aminotransferase (ALT) level was reduced to 44.88 ± 4.39 U/L, aspartate aminotransferase (AST) to 76.25 ± 4.17 U/L, alkaline phosphatase (ALP) to 60.75 ± 5.41 U/L, and acetaldehyde to 38.54 ± 1.01 U/L compared with that of the control group (ALT 72.38 ± 5.19 U/L, AST 119.63 ± 9.82 U/L, and ALP 98.63 ± 6.71 U/L and acetaldehyde 64.86 ± 4.70 U/L). QGHXR inhibited lipid overproduction and fibrotic gene expression. The serum concentration of chemokine C-X-C ligand 16 (CXCL16) was reduced to 62.83 ± 6.80 pg/ml compared with that of the control group (130.91 ± 13.72 pg/mL). QGHXR downregulated CXCL16 mRNA and protein expressions. Pharmacological CXCL16 treatment reversed the QGHXR-induced protective effects in ethanol plus CCl4 fed mice. QGHXR reduced CXCL16 levels (91.97 ± 5.86 pg/ml) in LPS-stimulated RAW264.7 cells compared with that of the control group (148.68 ± 8.62 pg/ml) and inhibited toll-like receptor 4 and nuclear factor-kappa B phosphorylation. Conclusions This study demonstrated that QGHXR mitigates experimental alcoholic liver fibrosis by CXCL16 inhibition, and may be considered a potential therapeutic agent for treating liver fibrosis.


Introduction
Liver fbrosis is a common pathological consequence of chronic liver diseases, including hepatitis B and C, alcohol abuse, and nonalcoholic steatohepatitis. It is characterized by excessive intercellular accumulation of extracellular matrix (ECM) in the liver [1]. Advanced liver fbrosis may progress to cirrhosis, which is responsible for liver failure and death [2]. High mortality associated with alcoholic liver fbrosis is caused by upper gastrointestinal bleeding, infection, and hepatic encephalopathy, which are attributed to alcohol-induced liver cirrhosis. Although research has been ongoing for many years to identify novel therapeutic targets for alcoholic liver disease (ALD), no medication has been approved by the FDA for the treatment of ALD [3]. Currently, the only efective treatment for liver fbrosis is liver transplantation [4]. Terefore, the development of novel antifbrotic agents for hepatic fbrosis is urgently required.
Alcoholic liver fbrosis is characterized by dyslipidemia, infammation, and fbrotic deposition. Inhibition of infammatory mediators, including chemokines, has been shown to ameliorate alcoholic liver fbrosis [5]. Chemokines, a family of small cytokines that can induce leukocyte migration, have been identifed as central regulators of liver fbrosis [6]. Chemokine C-X-C ligand 16 (CXCL16), which has soluble and transmembrane forms, is a ligand for the CXC chemokine receptor 6 (CXCR6) [7]. Several studies have emphasized the crucial role of CXCL16 in the progression of liver fbrosis [8][9][10]. CXCL16 is highly expressed in patients with hepatic fbrosis [9]. In addition, substantial antifbrotic efcacy has been observed in therapies that deactivate CXCL16 signaling [11]. Herein, we hypothesized that CXCL16 might be a critical target for alcoholic liver fbrosis.
As complementary and alternative medicine, traditional Chinese medicine (TCM) is receiving increasing attention worldwide for the prevention and treatment of various diseases [12,13]. TCM exhibits hepatoprotective efects against alcoholic liver fbrosis via heat-clearing and detoxifying efects, which are critical for antipyretic and antiinfammatory action, and dissipation of blood stasis in the theory of TCM [14]. Qinggan Huoxue recipe (QGHXR), which is composed of Radix bupleuri, Radix scutellariae, Radix salviae miltiorrhizae, Carapax trionycis, and Radix puerariae, is a traditional Chinese medicine that possesses prominent biological activities, including antiinfammatory, antiapoptotic, antisteatosis, and antifbrotic [15,16]. Te previous study showed that QGHXR prevents alcoholic liver fbrosis in rats by decreasing hepatocyte apoptosis and inhibiting epithelial-to-mesenchymal transition [15]. In addition, QGHXR showed benefcial efects and increased survival rates in rats with acute liver failure [17]. However, the molecular mechanism of the protective efect of QGHXR against alcoholic liver fbrosis remains elusive. Although CXCL16 exists as a crucial role in the progression of liver fbrosis, the role of CXCL16 in QGHXR-mediated antifbrotic efects has not yet been elucidated. Terefore, in this study, we treated mice with QGHXR to determine whether QGHXR could protect against ethanol + CCl 4 -induced liver fbrosis in a CXCL16-dependent manner.

Reagents and Drugs.
CCl 4 was purchased from Sigma-Aldrich (St. Louis, MO, USA). CXCL16 was purchased from PeproTech (Rocky Hill, NJ, USA). QGHXR is composed of Radix bupleuri 9 g, Radix scutellariae 9 g, Radix salviae miltiorrhizae 15 g, Carapax trionycis 9 g, and Radix puerariae 15 g. All herbs were obtained from Shanghai Sunbow Co. Ltd. (Shanghai, China) and authenticated by Associate Professor Yan Xi (Department of Pharmacy of Longhua Hospital). Te herbs were processed at the Department of Pharmacy of Longhua Hospital (Shanghai, China) to produce QGHXR.

Animals and Treatments.
Female C57BL/6J mice were obtained from Shanghai Sipper-BK Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed in a specifc pathogen-free environment at 23 ± 1°C with a 12 h light/dark cycle. All procedures were conducted according to protocols approved by the local ethics committee for animal research studies at the Shanghai University of Traditional Chinese Medicine. Female C57BL/6J mice were either fed a Lieber-DeCarli liquid diet containing 4% ethanol or pair-fed a control diet, and mice were intraperitoneally injected with a 2.5% solution of CCl 4 in olive oil (0.1 mL/kg) for 8 weeks, as previously described [18]. In week 5, the mice were administered 100, 200, and 400 mg/kg QGHXR via oral gavage daily for 4 weeks. Mice were divided into fve groups (

Qualitative Analysis Using Ultra Performance Liquid
Chromatography-Tandem Mass Spectrometry (UPLC-MS/ MS). UPLC-MS analysis was performed using a Shimadzu LCMS-8050 mass spectrometer coupled with a UPLC chromatograph. Chromatographic separation was achieved using a Shim-pack XR-ODS C18 column (2.0 × 75 mm, 1.6 μm). Te parameters used are as follows: capillary voltage, 15 kV; spray fow, 3 L/min; heating gas fow, 10 L/min; interface temperature, 350°C; DL temperature, 250°C; heating block temperature, 400°C; and drying airfow rate, 10 L/min. Gradient elution was performed using acetonitrile and water (2%-98%) for 120 min. Te MS spectra of the compounds were imported into the natural compound database for comparison, and the results with a similarity of more than 90% were taken for LCMS comparison analysis and qualitative analysis with the corresponding standards. Te LC-MS/MS chromatograms of the 45 compounds in QGHXR are shown in Figure 1.

Determination of Hepatic Lipid Levels.
Hepatic lipid levels were assessed according to the manufacturer's instructions of the relevant kits (Nanjing Jiancheng Bioengineering Institute).

Real-Time PCR.
Total RNA was extracted from the liver tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA using the PrimeScript RT kit (Takara). Real-time PCR was performed using the two-step quantitative RT-PCR method (Bio-Rad, Hercules, CA, USA). Te target gene expression was normalized to the average expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Te primers used are listed in Table 1.

Western Blot Assay.
To extract protein, the whole liver tissue was homogenized in ice-cold lysis bufer, as previously described [19]. Protein samples were subjected to SDS-PAGE and electroblotted to PVDF membranes (Bio-Rad). After blocking with 5% bovine serum albumin for 1 h, the membranes were incubated with primary antibody against CXCL16 or CXCR6 at 4°C overnight. Te membranes were washed in TBST (0.1% Tween) and incubated with the appropriate HRP-conjugated secondary antibody for 90 min at room temperature. Te signals were visualized using chemiluminescence and exposure to X-ray flms, and the relative expression level of the target protein was normalized to that of GAPDH. Fibrosis was assessed using Masson's trichrome staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer's protocol. Te sections were then visualized under a light microscope.

Hematoxylin and Eosin (H&E)
Staining. An H&E staining kit (Beyotime) was used for standard histological assessment. Samples were fxed in 4% paraformaldehyde, embedded in parafn, and cut into 5 μm sections. Te sections were then stained with H&E and observed under a light microscope.

Cell Culture.
Te murine macrophage cell line RAW264.7 was purchased from Shanghai Institute of Cell Biology (Shanghai, China) and maintained in Dulbecco's modifed Eagle's medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum in a 5% carbon dioxide atmosphere incubator at 37°C.

Statistical
Analysis. Data were analyzed using one-way analysis of variance (ANOVA) with SPSS (version 17.0; IBM, Chicago, IL, USA). Dunnett's multiple comparison post hoc test was used to determine group diferences. Data are expressed as mean ± SEM. Statistical signifcance was set at p < 0.05.

QGHXR Rescues Mice from Ethanol plus CCl 4 -Induced
Liver Injury. First, the composition of QGHXR was determined using UPLC-MS. Te chromatogram showed that QGHXR contained 45 compounds ( Figure 1). To determine   the efect of QGHXR on ethanol plus CCl 4 -induced liver injury, histological examination was conducted using H&E staining. Te levels of AST, ALT, ATP, and acetaldehyde were measured using the relevant biochemical kits. As indicated in Figure 2(a), treatment with QGHXR reduced ethanol plus CCl 4 -induced hepatocyte vacuolar degeneration and infammatory cell infltration (the arrows indicated the regions). QGHXR also improved several biochemical markers of liver injury, including serum AST, ALT, and ALP, and promoted hepatic acetaldehyde clearance (Figures 2(b) and 2(c)). Taken together, these results indicate that QGHXR administration attenuates ethanol plus CCl 4 -induced liver injury.

QGHXR Reduces Ethanol plus CCl 4 -Induced Hepatic
Steatosis. To examine whether QGHXR alleviates hepatic steatosis in vivo, hepatic lipid droplets and levels of triglycerides (TGs), free fatty acids (FFAs), and total cholesterol (CH) were evaluated in mice ( Figure 3). ORO staining showed more lipid droplets in ethanol plus CCl 4 -treated mice than in the pair-fed group, which were reduced with QGHXR treatment. Biochemical lipid measurements demonstrated severe TG and FFA deposition in the livers of ethanol + CCl 4 model mice relative to pair-fed animals, which was markedly attenuated by QGHXR administration. However, no signifcant diference was detected in the hepatic CH levels. Collectively, these data suggest that QGHXR protects mice from ethanol plus CCl 4 -induced hepatic steatosis.

QGHXR Decreases CXCL16 Expression in Ethanol plus
CCl 4 -Induced Liver Fibrosis. To explore the mechanisms underlying the benefcial efects of QGHXR on liver fbrosis, we studied the CXCL16/CXCR6 axis in ethanol plus CCl 4treated mice following QGHXR administration. Ethanol plus CCl 4 treatment resulted in an increase in serum CXCL16 concentration ( Figure 5(a)). Consistent with the ELISA data, the mRNA and protein levels of CXCL16 were elevated in the livers of ethanol plus CCl 4 -treated animals (Figures 5(b) and 5(c)). Despite the increase in CXCL16 expression, no change was observed in the CXCR6 levels. It is noteworthy that QGHXR administration profoundly reversed serum CXCL16 levels, and similar reductions were observed in the liver CXCL16 mRNA and protein levels. Taken together, these results suggest that CXCL16 signaling is involved in the QGHXR-induced protective efects in experimental alcoholic liver fbrosis.

CXCL16 Treatment Reversed QGHXR-Induced Protective Efects in Ethanol plus CCl 4 -Treated Mice.
To confrm the role of CXCL16 in QGHXR-induced protective efects in experimental liver fbrosis, pharmacological CXCL16 treatment was used in this study. As shown in Figure 6, CXCL16 treatment abolished QGHXR-induced attenuation of liver injury, as evidenced by increased liver injury in H&E-stained liver sections and elevated serum AST, ALT, and ALP production and hepatic acetaldehyde in the ethanol + CCl 4 + CXCL16 + 400 group when compared with those of the ethanol + CCl 4 + 400 group. In addition, hepatic TG and FFA levels were markedly depleted after QGHXR treatment alone but increased by CXCL16 and QGHXR cotreatment, suggesting that CXCL16 treatment suppressed QGHXR-induced efects on hepatic steatosis (Figure 7). No diference was found in the hepatic CH levels. Ten, we focused on the infuence of CXCL16 treatment on the QGHXR-induced protective efects in liver fbrosis. As shown in Figure 8, collagen accumulation, fbrotic gene expression (α-SMA, Collagen1α1, TIMP-1, and TGF-β), and hydroxyproline levels were inhibited by QGHXR administration and reversed by CXCL16 treatment. Based on the abovementioned fndings, CXCL16 inhibition is required in QGHXR-exerted antifbrotic efects.

QGHXR Inhibits Toll-like Receptor 4 (TLR4) and Phosphorylated Nuclear Factor-Kappa B (p-NF-κB) Expression In
Vitro and In Vivo. According to a previous study [10], Western blot assay shows that QGHXR also inhibited TLR4 and p-NF-κB levels in RAW264.7 cells (Figure 9(c)). To confrm the efect of QGHXR on the TLR4/NF-κB pathway, TLR4 and p-NF-κB expression levels were determined in the mouse liver. In line with the in vitro results, QGHXR also remarkably inhibited TLR4 and p-NF-κB expression in the ethanol plus CCl 4 -administrated mouse liver. Tese results indicate that the TLR4/NF-κB pathway is responsible for the inhibition of CXCL16 by QGHXR.

Discussion
Qinggan Huoxue recipe (QGHXR) is a Chinese herbal prescription that exerts benefcial efects on liver fbrosis. In this study, we explored the antifbrotic efects of QGHXR. We found that QGHXR alleviated liver injury, hepatic steatosis, and hepatic fbrosis and downregulated CXCL16 expression in ethanol plus CCl 4 -induced liver fbrosis. Furthermore, pharmacological CXCL16 administration ablated the QGHXR-induced protective efects in experimental alcoholic liver fbrosis.
Ethanol feeding and CCl 4 injection-induced murine models have been widely used to study liver fbrosis [18,20]. CCl 4 is a hepatotoxic chemical that efectively induces liver fbrosis in rodents, and ethanol exposure was shown to accelerate CCl 4 -induced liver fbrosis [21,22]. Liver damage, including hepatocyte necrosis, steatosis, and fbrosis, has been reported in ethanol plus CCl 4 -treated animals [20,23]. In this study, mice that underwent ethanol feeding and CCl 4    Evidence-Based Complementary and Alternative Medicine infammatory cell infltration. UPLC-MS/MS result showed that QGHXR contained 45 compounds including salvianolic acid A, scutellarin, baicalin, rutin, and chai saponin D. Te efects of these natural products in QGHXR on liver disease were extensively reported. Salvianolic acid A suppressed CCl4-induced liver fbrosis in mice by inhibiting infammation and oxidative stress [24]. Scutellarin protected against CCl 4 -liver injury in mice by repressing CYP2E1 and IκBα/NF-κB signaling pathways and modulating the gut microbiota [25]. Baicalin ameliorates alcohol-induced hepatic steatosis by suppressing SREBP1celicited PNPLA3 competitive binding to ATGL [26]. With a multi-ingredient and multitarget-pathway pharmacological action, QGHXR as a traditional Chinese medicine is compatible with the complex pathogenesis of ALD to mitigate it. It was believed that the integrated regulation of QGHXR makes it more competitive than any other chemical drugs or active ingredients.  Evidence-Based Complementary and Alternative Medicine Chemokines are involved in the development of steatosis, infammation, and fbrosis in alcoholic liver disease [27]. Tus, it has been hypothesized that inhibition of chemokines is the key mechanism of QGHXR-modulated alcoholic liver fbrosis. CXCL16, a small-inducible cytokine B16 and SR-PSOX, belongs to the type I membrane proteins of the CXC chemokine family. CXCL16 is a multifunctional chemokine that combines the scavenger receptor function with the properties of infammatory chemokines [28]. CXCL16 is universally believed to serve a pivotal molecule that controls infammatory and immune reactions. CXCL16 reduces local infammation and drives chemokinedependent activation of leukocytes in the liver. CXCR6, the receptor of CXCL16, is implicated in multiple infammatory dysfunctions. Previous studies suggested that CXCL16 plays a vital role in liver disorders. CXCL16 exists     on the surface of antigen-presenting cells and hepatocytes in patients with hepatic disorders [29]. Hepatic CXCL16 mRNA and protein levels are increased in gallstones accompanied by liver injury [30]. Te administration of an anti-CXCL16 antibody increased the survival of mice with immunological liver injury by reducing the infltration of T lymphocytes into the liver tissue [31]. Activation of CXCL16 signaling enhances lipid deposition in fatty livers of apolipoprotein E knockout mice [32]. Te serum concentration of CXCL16 is positively correlated with the activities of ALT and AST in patients with gallstones [7]. More severe hepatic pathological alterations have been observed in CXCL16defcient mice [29]. Te above evidence indicates that CXCL16 may play a critical role controlling liver repair in QGHXR-treated mice. Our results confrmed that QGHXR administration attenuated histopathological changes, inhibited transaminase activity, and suppressed dyslipidemia.
CXCL16 also plays a key role in liver fbrosis. Increased hepatic CXCL16 mRNA expression was found in patients with liver fbrosis compared to controls [10]. NKT cells drive hematopoietic stem cell activation and liver fbrosis by stimulating CXCL16 in chronic liver injury [10]. CXCL16 and CXCR6 have been proposed to be responsible for anchoring activated NKT cells [10]. Metformin, a frst-line agent for type 2 diabetes treatment, was demonstrated to reduce the gene expression levels of several fbrotic markers, including α-SMA, Collagen1α1, and TGF-β1, which is associated with the reduction of CXCL16 mRNA expression [11]. Te current study showed that ethanol feeding and CCl 4 injection resulted in increased serum CXCL16 concentrations and upregulated hepatic CXCL16 mRNA and protein expression, suggesting the involvement of CXCL16 in the development of liver fbrosis. Importantly, it has been reported that in the murine liver, CXCL16 is strongly expressed by the endothelium and macrophages, whereas lymphocytes, including NKT, NK, CD4, and CD8 T cells, express CXCR6 [10]. In the present study, we found that QGHXR did not alter CXCR6 levels, indicating that lymphocytes may not be the target cells of QGHXR. Moreover, excessive alcohol intake impairs the intestinal barrier and increases intestinal permeability, resulting in endotoxins entering the portal venous circulation, which in turn induces proinfammatory cytokine and chemokine production in Kupfer cells [33]. QGHXR inhibited endotoxin-induced CXCL16 levels and blocked the TLR4/NF-κB pathway in macrophages ( Figure 9). Consistent with the in vitro results, QGHXR intervention robustly decreased CXCL16 levels in the serum and liver of ethanol-fed mice injected with CCl 4 , indicating that the reduced CXCL16 level is partially attributed to QGHXR-induced protective efects in experimental liver fbrosis. Moreover, pharmacological CXCL16 treatment abolished QGHXR-induced benefcial efects in the ethanol plus CCl 4 mouse model, as evidenced by liver function biomarkers, dyslipidemia, and fbrotic indices. Tese results further demonstrate that CXCL16 deactivation is required for the antifbrotic efcacy of QGHXR.

Conclusions
Tis study demonstrates that QGHXR protects mice from ethanol plus CCl 4 -induced liver fbrosis. Te protective properties of QGHXR are dependent on CXCL16 deactivation. Our results reveal the potential mechanism underlying QGHXR-induced antifbrotic efects and suggest that CXCL16 could serve as a novel target for the treatment of liver fbrosis.

Data Availability
Te data used to support the fndings of this study are available from the corresponding authors upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.