Astragali Radix Contributes to the Inhibition of Liver Fibrosis via High-Mobility Group Box 1-Mediated Inflammatory Signaling Pathway

Astragali Radix (AR), the dried root of Astragali Radix membranaceus (Fisch.) Bge. or Astragali Radix membranaceus (Fisch.) Bge. var. mongholicus (Bge) Hsiao, is a commonly used traditional Chinese medicine for the treatment of liver diseases. This study aimed to comprehensively evaluate the pharmacological action and explore the potential mechanism of AR on liver fibrosis. Rats were administered with carbon tetrachloride for eight weeks, followed by oral treatment with AR for six weeks. The efficacy was confirmed by measuring liver function and liver fibrosis levels. The underlying mechanisms were explored by detecting the expression of related proteins. AR significantly decreased the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), collagen IV (COL-IV), hyaluronic acid (HA), laminin (LN), and precollagen type III (PCIII). In addition, AR inhibited the deposition of collagen and the activation of hepatic stellate cells. Those data strongly demonstrated that AR alleviated liver fibrosis by CCl4. In order to illustrate the potential inflammatory, the mRNA levels of IL-6, TNF-α, and IL-1β were detected. Subsequently, immunohistochemistry analysis was performed to further verify the expression of type I collagen. Finally, the expression of key proteins in the inflammatory signaling pathway was detected. AR significantly reduced the expression of high-mobility group box 1 (HMGB1), TLR4, Myd88, RAGE, and NF-κ B p65 genes and proteins. In addition, western blotting showed AR decreased the protein expression of RAGE, p-MEK1/2, p-ERK1/2, and p-c-Jun. Taken together, our data reveal that AR significantly inhibits liver fibrosis by intervening in the HMGB1-mediated inflammatory signaling pathway and secretion signaling pathway. This study will provide valuable references for the in-depth research and development of Astragali Radix against liver fibrosis.


Introduction
Liver fibrosis is a complex pathological process and an intermediate link between various liver diseases including cirrhosis and liver cancer [1,2]. And prevention of malignant liver diseases by inhibiting the occurrence and development of liver fibrosis has attracted worldwide attention [3]. Numerous studies have revealed the pathogenesis of liver fibrosis, such as inflammatory response, activation of hepatic stellate cells, and excessive deposition of extracellular matrix, but it has not yet been transformed into an effective and powerful treatment [4,5]. ere is still a lack of safe and effective means of treating liver fibrosis [6,7].
It is necessary to develop new drugs for treating liver fibrosis [7]. Traditional Chinese medicine has potential advantages in the treatment of chronic diseases, so we tried to find effective drugs for the treatment of liver fibrosis. Astragali Radix (AR), the dried root of Astragali Radix membranaceus (Fisch.) Bge. or Astragali Radix membranaceus (Fisch.) Bge. var. mongholicus (Bge) Hsiao, is a commonly used traditional Chinese medicine for the treatment of liver diseases [8]. And it has been proved that AR possesses hepatoprotective and anti-inflammatory properties and has been shown to exhibit immunomodulating and antioxidant activity, among others [9]. In traditional Chinese formula, AR is often used for the treatment of liver fibrosis [10]. Although the protective effects of AR on liver injury and related mechanisms have been reported [9,11], the studies on the effect and mechanism of AR on liver fibrosis are still lacking. More importantly, toxicological study has confirmed the high safety of AR. No significant toxicity or side effects were observed when the dose administered to rats was set to 39.9 g/kg [12].
Experimental and clinical studies have found that the high-mobility group box-1 (HMGB1), recently discovered damage-associated molecular patterns (DAMPs), is substantially enriched in patients with hepatic fibrosis [13,14]. HMGB1 is a late inflammatory mediator that plays an important role in the pathogenesis of many chronic inflammations, which is the main cause of liver fibrosis [15].
ere is evidence that HMGB1 can directly stimulate hepatic stellate cells (HSCs) activation to accelerate the development of liver fibrosis [16]. More importantly, toll-like receptor 4 (TLR4) and the receptor of advanced glycation end products (RAGE) are the main receptors of HMGB1 [15]. It is reported that the binding of extracellular HMGB1 to TLR4 could activate NF-κB signaling to induce inflammation and aggravate liver fibrosis. Studies have shown that extracellular HMGB1 binding to RAGE could phosphorylate c-Jun to induce a significant increase in type I collagen secretion [16,17], which directly accelerates the development of liver fibrosis (c-Jun is a component of the heterodimeric AP1 and is a potent transactivating factor for the collagen1a1 and collagen1a2 genes [17,18]).
Based on previous studies, this study aims to provide answers to the following questions: Does AR have therapeutic effect on carbon tetrachloride-(CCl 4 -) induced liver fibrosis in rats? What is the underlying mechanism? Is the mechanism related to downregulation of HMGB1? e animals were maintained under controlled conditions of temperature 25°C ± 2°C, humidity 55% ± 5%, and with light and dark cycles.

Materials and Methods
All the animals were randomly divided into six groups with six rats in each group. Olive oil diluted CCl 4 in a ratio of 1 :1. e model group, AR (2.7, 5.4, 10.8 g/kg/d) groups, and colchicine (as a positive control group) groups were given CCl 4 diluent (2 mL/kg) twice a week for 8 weeks [19]. At the same time, the control group was intraperitoneally injected with the same dose of olive oil. Eight weeks later, the AR (2.7, 5.4, and 10.8 g/kg/d) groups and colchicine group (0.2 mg/kg/d) were given the corresponding concentration of drugs [20]. e control group and model group were given equal volume of distilled water. All rats were treated by intragastric administration once a day for 6 weeks. All rats were deprived of food for 24 h before the last treatment but allowed free access to water. e animals were sacrificed 2 h after the last treatment and their liver and serum were harvested for the further studies. e blood samples were centrifuged at 3500 rpm for 15 min. e liver tissue was divided into 3 pieces and placed in the sample bottle. Serum and liver tissue were stored at −80°C.
All animal experiments were approved by the Ethical Committee of the Fifth Medical Center of Chinese PLA General Hospital in China.

Biochemical Assay.
e serum levels of ALT, AST, COL-IV, HA, LN, and PC III were measured. In brief, according to the manufacturer's protocols, the samples, 2,4-dinitrophenylhydrazine solution, and sodium hydroxide solution were added in turn. e final color was read at 510 nm, and the serum ALT and AST levels were determined according to the OD value. Similarly, according to the manufacturer's instructions, add the reagents in the kit in turn, read the final color at 450 nm, and obtain the serum levels of COL-IV, HA, LN, and PC III according to the OD value.

Histological Examination.
Liver tissue was fixed in 10% neutral buffered formalin for more than 48 h. All the livers were eluted with gradient ethanol solution and fixed and embedded in paraffin. Tissue sections about 4-5 μm in thickness were cut and stained with hematoxylin eosin (H&E) and Masson. e pathological changes in the liver tissues were observed under a Nikon microscope (Nikon Instruments Inc., Japan).

Quantitative Reverse Transcription Polymerase Chain
Reaction (RT-PCR) Analyses. Quantitative RT-PCR was used to detect the mRNA expression of IL-6, TNF-α, IL-1β, HMGB1, TLR4, Myd88, and RAGE in liver tissues. Total RNA was extracted from 90 mg frozen liver tissue by Trizol reagent (Life Technologies, CA, USA). e absorbance of the extracted mRNA was detected by spectrophotometer at 260 nm and 280 nm, respectively. e mRNA with purity (A260/A280) between 1.8 and 2.2 was selected as the qualified product for the follow-up study. RNA (2 μg) was transcribed into cDNA by PrimerScript RT regent kit ( ermo Fisher Scientific, MA, USA). e cDNA was subsequently subjected to PCR amplification by ABI 7500 Real-Time PCR machine (Applied Biosystems Inc, Carlsbad, CA, USA). Data analysis was performed by the 2 −△△CT method. e primers are listed in Table 1.

Western Blotting.
e mixture of ice-cold radio immunoprecipitation assay (RIPA) lysis buffer, phenylmethylsulfonyl fluoride (PMSF), and protein phosphatase inhibitor was used for protein extraction. Liver tissue (80 mg) was homogenized and lysed in the mixture, and then centrifuged at 12000 × g and 4°C for 10 min. e supernatant was collected and BCA Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China) was used to detect the total protein concentration. All protein samples were levelled to the same concentration. en, reducing Laemmli SDS sample buffer was added and mixed well, and boiled in boiling water for 5 minutes. e prepared samples were western blotted with 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the blot was transferred to the polyvinylidene fluoride (PVDF) membrane. e PVDF membrane was sealed in 5% skimmed milk at room temperature for 2 hours and then placed in a mixture of 5% skimmed milk, Tris-buffered saline (TBS), and 0.05% Tween 20 with the corresponding primary antibody, and incubated overnight at 4°C. Antibodies, including rabbit Anti-NF-κB p65 (Cell Signaling Technology, dilution: 1 : 1000), RAGE (ABclonal technology, dilution: 1 : 1000), phospho-MEK1/2 (Cell Signaling Technology, dilution: 1 : 2000), MEK1/2 (Abcam, dilution: 1 : 10000), phospho-ERK1/ 2 (Cell Signaling Technology, dilution: 1 : 1000), ERK1/2 (Cell Signaling Technology, dilution: 1 : 10000), phospho-c-Jun (Abcam, dilution: 1 : 500), and GAPDH (ABclonal technology, dilution: 1 : 10000), were used at this step. GAPDH was used as control for total protein extract. After incubation with appropriate secondary antibody for 2 hours at room temperature, the membrane was washed 3 times in TBS with 0.05% Tween 20 for 5 minutes each. Finally, the chemiluminescence system was used to measure immunoreactive proteins. All the western blotting was repeated 3 times.

Immunohistochemistry Analysis.
To evaluate the effects of AR on the expression of α-SMA, HMGB1, and collagen I in liver tissue of fibrosis rats, immunohistochemical analysis was performed as described previously [21]. Paraffin sections of liver tissues were put in xylene and gradient ethanol for deparaffinization and dehydration, and then the sections were put into citric acid antigen retrieval solution for antigen retrieval. e sections were treated with 3% hydrogen peroxide solution to block endogenous peroxidase. e sections were then blocked with 1% BSA for 30 minutes at room temperature. Subsequently, the sections were incubated with α-SMA (Cell Signaling Technology, dilution: 1 :100), Rabbit Anti-HMGB1 (Cell Signaling Technology, dilution: 1 : 200), and collagen type I (Abcam, dilution: 1 : 200) in a PBS solution at 4°C overnight. After incubating the sections with appropriate secondary antibodies for 1 hour at room temperature, they were stained with DAB staining solution and hematoxylin, and finally the slides were dehydrated and mounted. Nikon microscope (Nikon Instruments Inc., Japan) and NIS-Elements (F 4.0 version, Japan) software were used to observe the sections. Finally, Image-Pro Plus (version 6.0, Media Cybemetics, INC., Rockville, MD, USA) software was used to calculate and analyze the average integrated optical density (IOD) of the positive area of the immunohistochemical reaction image.

Statistical Analysis.
Results were expressed as mean-± standard deviation (SD). Data were evaluated by one-way ANOVA and Duncan's multirange test with the SPSS computer program (version 20.0, SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant and P < 0.01 was considered highly significant.

AR Has erapeutic Effect on CCl 4 -Induced Liver Injury.
As shown in Figures 1(a) and 1(b), the levels of ALT and AST in rats in the model group were significantly increased (P < 0.01) compared with those in the control group. And serum ALT and AST concentrations were significantly reduced after oral administration of AR 5.4 g/kg and 10.8 g/kg (P < 0.01). Subsequently, the serum levels of COL-IV, HA, LN, and PC III which could reflect the degree of hepatic fibrosis were examined [22]. As shown in Figures 1(c)-1(f), the serum levels of COL-IV, HA, LN, and PC III in the model group were significantly higher than those in the control group (P < 0.01). e inhibitory effect of AR on the levels of COL-IV, HA, LN, and PC III is dose-dependent. In particular, the serum levels of COL-IV, HA, LN, and PC III were significantly decreased in the 10.8 g/kg AR group (P < 0.01).

e Effect of AR on Liver Histopathological Changes and Liver Fibrosis.
en, hematoxylin-eosin (HE) staining and Masson staining were used to observe the pathological changes of liver tissue [21,23]. e results of HE showed that the liver tissue structure of the control group was normal without morphological changes. e long-term CCl 4 -treated rats showed partial necrotic in their liver accompanied by severe steatosis and inflammatory cell infiltration around the central vein of hepatic lobules. In contrast, the degree of liver damage, steatosis, and inflammatory infiltration in the AR treatment groups were reduced to varied degrees in a dosedependent manner (Figure 2(a)). In the liver section of Masson staining, the blue area represented the deposited collagen. e results show that a large amount of collagen was deposited around the hepatic sinus of the model group. AR can effectively reduce collagen deposition and improve liver fibrosis (Figure 2(b)). e above results confirmed that the CCl 4 -induced rat liver fibrosis model was successful, and AR could effectively ameliorate liver fibrosis in rats.

Inflammatory Response in Rats Induced by CCl 4 .
Clinical data suggest that inflammation is a key factor in the progression of liver fibrosis [24]. IL-6, as a classic proinflammatory cytokine biomarker, is used for clinical diagnosis of chronic liver fibrosis [25]. TNF-α can promote the survival of activated HSCs and promote the development of liver fibrosis [26]. It is reported that the serum IL-1β in patients with liver fibrosis caused by schistosomiasis is significantly increased [24]. erefore, we detected the expression of IL-6, TNF-α, and IL-1β in rats by quantitative RT-PCR. e mRNA levels of IL-6, TNF-α, and IL-1β were increased remarkably in   -3′)  IL-6  AGGAGTGGCTAAGGACCAAGACC  TGCCGAGTAGACCTCATAGTGACC  TNF-α  GCATGATCCGAGATGTGGAACTGG  CGCCACGAGCAGGAATGAGAAG  IL-1β  ATCTCACAGCAGCATCTCGACAAG  CACACTAGCAGGTCGTCATCATCC  HMGB1  ACAACACTGCTGCGGATGACAAG  CCTCCTCGTCGTCTTCCTCTTCC  TLR4  GCTGCCAACATCATCCAGGAAGG  TGATGCCAGAGCGGCTACTCAG  Myd88  CGACGCCTTCATCTGCTACTGC  CCACCACCATGCGACGACAC  RAGE  CTGCCTCTGAACTCACAGCCAATG  GTGCCTCCTGGTCTCCTCCTTC  GAPDH  GTCCATGCCATCACTGCCACTC  GATGACCTTGCCCACAGCCTTG   4 Evidence-Based Complementary and Alternative Medicine the CCl 4 -induced group compared with the control group (P < 0.01, Figure 3). After treatment with AR, the expression of related genes decreased in a dose-dependent manner (P < 0.05, P < 0.01). e results showed that AR had therapeutic effect on CCl 4 -induced liver inflammation in rats.

AR Downregulates CCl 4 -Induced Liver Inflammation.
Literature research found that HMGB1, a cytokine abundantly enriched in patients with liver fibrosis, mediated TLR4//NF-κB signaling pathway is one of the important pathways for liver aseptic inflammation. erefore, we tested the expression levels of HMGB1, TLR4, Myd88, and NF-κB p65, which are the key targets of this signaling pathway in liver fibrosis rats. Quantitative RT-PCR and immunohistochemistry (IHC) methods were used to detect the expression and subcellular localization of HMGB1. As shown in Figures 4(a)-4(e), in the control group, HMGB1 was mainly located in the nucleus of hepatocytes and rarely in the cytoplasm or sinusoids. In the model group, the greater HMGB1 immunoreactivity was observed in the periportal and bridge fibrosis areas in the liver. In addition, computer-assisted semiquantitative analysis showed that AR treatment alleviated the expression of HMGB1 compared with the model group (Figure 4(f ), P < 0.05, P < 0.01).
Furthermore, the mRNA expression of HMGB1 was also quantified by using the RT-PCR method. e results showed that high dose of AR could significantly reduce the mRNA expression of HMGB1, which is consistent with the result of the IHC method. ose results proved that AR could reduce the expression of HMGB1 (Figure 4(g)). Subsequently, quantitative RT-PCR was used to detect the TLR4, Myd88, and RAGE mRNA levels in the model group, which were significantly higher than those in the control group (Figures 4(h)-4(j), P < 0.01). After treatment with AR, the expression of related genes decreased in a dose-dependent manner (P < 0.05, P < 0.01). Western blotting was used to significantly thicker than that of the control group, and the statistical results showed significant differences (P < 0.01). After AR treatment, the expression of NF-κB p65 decreased in   Evidence-Based Complementary and Alternative Medicine a dose-dependent manner, especially in the 5.4 g/kg AR group and 10.8 g/kg AR group (P < 0.01). ese results suggest that AR may improve liver inflammation by downregulating HMGB1/TLR4//NF-κB signaling pathway.

AR Inhibits the Activation of HSCs.
HSCs are the core cells of liver fibrosis. It is reported that HMGB1 can directly activate HSCs [16]. erefore, we speculate that AR can play a protective role in liver fibrosis by reducing the activation of HSCs. α-smooth muscle actin (α-SMA) is a special marker of activated HSCs [27]. Here, the ratio of activated HSCs was measured by using IHC method through detecting α-SMA in the liver tissues ( Figure 5). In the control group, only a small amount of α-SMA-positive cells was found in the portal area, which might be the hepatic arteries. More α-SMA-positive cells were observed in the CCl 4 -induced group compared with control group, and the α-SMA-positive cells were significantly reduced after AR treatment. By computer-assisted semiquantitative analysis, the mean integrated optical density (IOD) of the α-SMA-positive area of the AR treatment group was confirmed to be significantly reduced in a dose-dependent manner compared to model group ( Figure 5, P < 0.01). ose results revealed that AR could reduce the activation of HSCs, a key cell of liver fibrosis.

AR Has an Effect on the Type 1 Collagen Secretion Signaling
Pathway Mediated by HMGB1. Next, IHC method was used to detect the expression of collagen type I. In Figures 6(a)-6(e), type I collagen deposition around the hepatic lobular portal vein in rats treated with CCl 4 alone was significantly increased. After treatment with AR, the expression and deposition of type I collagen in the liver were significantly inhibited, especially in the group with the dose of 10.8 g/kg. e semiquantitative analysis of type I collagen immunopositive region further confirmed the above results ( Figure 6(f )). e mean IOD of type I collagen in the AR administered group was significantly reduced in a dosedependent manner compared to the model group (P < 0.01). It was previously reported that HMGB1 interacted with the receptor of RAGE to regulate the expression of type I collagen [17]. erefore, we explored the effects of AR on the expression of downstream targets of HMGB1, such as RAGE, p-MEK1/2, p-ERK1/2, p-cJun, and type I collagen. Western blot method was used to detect the expression of RAGE, p-MEK1/2, p-ERK1/2, and p-cJun protein after administration of AR (Figures 6(g)-6(k)). e proteins of RAGE, p-MEK1/2, p-ERK1/2, and p-cJun were increased remarkably only in the CCl 4 -induced group compared with the control group (P < 0.05, P < 0.01). Compared with the model group, the high-dose administration group of AR significantly reduced the expression of RAGE, p-MEK1/2, p-ERK1/2, and p-cJun (Figures 6(h)-6(k), P < 0.05, P < 0.01).

Discussion
In this study, the aqueous extract of AR was used, which is consistent with the clinical use of traditional Chinese medicine. And the research results are more valuable. To determine whether AR has a protective effect on liver fibrosis, CCl 4 -induced liver fibrosis model was performed in rats. Consistent with clinical observations, AR could improve liver function to some extent to alleviate liver injury [28,29]. In this study, the results demonstrated that AR could reduce serum markers of liver fibrosis, including COL-IV, HA, LN, and PC-III. e examination of serum levels and liver histopathology exhibited that AR showed obvious anti-liver fibrosis effect and the anti-liver fibrosis effect of AR at the dose of 10.8 g/kg is comparable to that of colchicine. In order to apply AR more scientifically, the potential mechanism of AR on alleviating liver fibrosis was further explored.
Previous reports and the results of this study confirmed that AR has definite hepatoprotective effect and can effectively improve liver fibrosis [19,30,31]. However, how to exert the therapeutic effect is still unclear. Previous studies have reported that AR has strong anti-inflammatory activity and can effectively reduce the expression of inflammatory factors such as iNOS, cyclooxygenase-2 (COX-2), IL-6, TNF-α, and IL-1β [32,33]. Long-term inflammatory response is the key to the occurrence of liver fibrosis [34]. erefore, we detected the effect of AR on the expression of inflammatory factors in liver fibrosis rats. e results showed that AR could significantly downregulate the expression of IL-1β, TNF-α, and IL-6. It suggested that AR might improve liver fibrosis by inhibiting the inflammatory response in rats.
Pathogen-free inflammatory liver disease is considered "sterile" [35]. Studies have reported that DAMP is the key to aseptic inflammation after liver injury, and HMGB1 is the key point [36]. Extracellular HMGB1 is a multifunctional cytokine, which induces inflammation by binding with specific cell surface receptor TLR4 [37]. us, we detected the expression of HMGB1 in rats to explore whether AR had effect on regulating HMGB1 expression via improving the inflammatory response. e results showed that the expression of HMGB1 decreased in the AR treatment group compared with the model group.
e TLR4 signaling pathway is the most studied pathway. TLR4, as a pattern recognition receptor, recruits Myd88 under the action of the endogenous activator HMGB1, which causes the activation of the NF-κB inflammatory pathway and promotes the transcription of inflammatory cytokines IL-1β, TNF-α, and IL-6 [38,39]. erefore, we detected the expression of TLR4, Myd88, and NF-κB p65. e results showed that the expression levels of TLR4, Myd88, and NF-κB p65 in AR treatment group were significantly lower than those in the model group. e above results suggest that AR might reduce CCl 4 -induced liver inflammatory by downregulating the expression of HMGB1/TLR4/NF-κB signaling pathway.
In experimental and human liver injury, activated HSCs (myofibroblasts) are recognized as the driving factors of liver fibrosis [40]. It has been reported that HSCs activated by inflammatory factors are the main source of myofibroblasts in the liver [34]. And HMGB1 could directly stimulate the activation of HSCs [16]. e pharmacodynamic results show that AR can effectively improve the liver fibrosis in rats induced by CCl 4 . e study also found that AR can not only   downregulate the expression of HMGB1, but also reduce the transcription of inflammatory cytokine IL-1β, TNF-α, and IL-6 by regulating HMGB1/TLR4/NF-xB signaling pathway. erefore, we speculate that AR can inhibit the activation of rat HSCs. e detection of α-SMA, a marker protein of HSCs activation, further confirmed our hypothesis: AR can significantly inhibit the activation of rat HSCs induced by CCl 4 (Figure 7).
Liver fibrosis or scar deposition caused by chronic injury is similar in all forms of liver disease [41]. In the subendothelial space between hepatocytes and endothelial cells, fibrillar or type I collagen aggregates to replace the lowdensity basement membrane-like matrix containing type IV collagen. e conversion of the subendothelial matrix to fibril-rich collagen is a key event, which mediates the loss of differentiation function characteristic of progressive liver disease [42]. Myofibroblasts are the main source of type I collagen accumulated during tissue fibrosis [43]. It was found that the activation of HSCs was decreased in ARtreated group, and we speculated that the expression of type I collagen in the liver of rats would also be reduced accordingly. In addition, the latest research reports that HMGB1 activates the paracrine system and upregulates the secretion of type 1 collagen by binding to the receptor RAGE on the surface of fibroblasts, thereby triggering and/or maintaining scar formation [17]. erefore, we detected the expression of type I collagen in the liver of rats, as well as the expression of RAGE, p-MEK1/2, p-ERK1/2, and p-c-Jun in liver tissues. e results showed that AR can significantly reduce the expression of type 1 collagen in rats, which may be related to the decrease of HSCs activation and the downregulation of collagen secretion mediated by HMGB1-RAGE-c-Jun.

Conclusion
In conclusion, biochemical tests and histological assessments were used to reveal the function of AR. e study also demonstrated that AR was potent for downregulating the expression of HMGB1/RAGE/cJun pathway and HMGB1/ TLR4/NF-κB pathway, thus protecting rats against CCl 4 -  Evidence-Based Complementary and Alternative Medicine 9 induced liver fibrosis. erefore, this study will provide scientific evidence and theoretical basis for the development of AR as a potential drug candidate for liver fibrosis.