The extract of a Traditional Chinese Medicine alleviates Hepatic Fibrosis of Rats by inhibiting NF-κB activation and upregulating BAMBI in Hepatic Stellate Cells

mobility shift assay; ECM: extracellular matrix; TGF-β: Transforming growth factor-β ; LPS :Lipopolysaccharide ; NF-κB :nuclear factor-κB; TCM: traditional Chinese medicine; H&E :hematoxylineosin ; NPC: Non-parenchymal cell; PBS:phosphate buffered saline.


Abstract
Background DaHuangZheChong pill (DHZCP) is a formula of traditional Chinese medicine, which has been written into the guideline for the prevention and treatment of hepatic brosis in China. The study was aim to investigate the anti-brotic effects and the potential mechanisms of DHZCP revolving around the TGF-β pseudo receptor, bone morphogenic protein and activin membrane-bound inhibitor (BAMBI) in hepatic stellate cells (HSCs).

Materials and Methods
Wistar rats were given with CCL4 for four weeks to establish hepatic brosis model. Then the rats were given normal saline or DHZCP decoction six weeks. the pathology of liver tissue was analyzed, the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), BAMBI, and NF-κB were detected. In vitro, the associated signal molecules about LPS-activated NF-κB were also analyzed by immunohistochemistry, western blot, or electrophoretic mobility shift assay (EMSA) in cultured HSC-T6 cells .

Results
The DHZCP showed signi cant effects on improving brosis stage of liver tissue and inhibiting primary HSCs activation. The protein expression of TLR4/MyD88 was lower (P was both < 0.05), BAMBI was higher in DHZCP group than model control (MC) group (P < 0.05) in primary HSCs. In HSC-T6 cells, the activity of NF-κB was lower (P < 0.001), and BAMBI was higher (P < 0.05) in DHZCP added LPS group than in LPS group.

Conclusion
These results suggested that DHZCP alleviates hepatic brosis that was maybe associated with inhibiting activation of NF-κB induced by LPS, and upregulating BAMBI expression in HSCs.

Background
Chronic liver disease is often accompanied by persistent liver damage such as chronic viral infections, widespread alcohol abuse,fatty liver diseases and cholestasis. The liver injuries contribute to a woundhealing response, excessive deposition of extracellular matrix (ECM) and reconstruction of tissue structure resulting in liver brosis [1].
Hepatic stellate cells (HSCs) were essential to the occurrence and development of liver brosis.
Hepatic brosis alleviation and cirrhosis degradation have been reported in recent years [5,6], clinical and experimental evidence also revealed hepatic liver could be reversible [7,8]. Anti-brosis has become an indispensable step in the development of chronic liver disease. Chinese herbal medicine has the pharmacological characteristics of multi-pathway, multi-level and multi-target to alleviate hepatic brosis [9,10]. DaHuangZheChong pill (DHZCP) was a classic recipe in traditional Chinese medicine(TCM), and was o cially approved and recommended for treat liver brosis [11]. Many clinical and experimental studies had con rmed the effect on hepatic brosis [12,13], and de nited that the anti-brotic mechanism was about inhibiting the activation of HSCs [14]. However, it was not enough to elucidate the mechanism for treating hepatic brosis.
We hypothesized that the possible anti-brosis mechanism of DHZCP was to suppress the activation of NF-κB by LPS, and increas the expression of BAMBI in HSCs.

Animal experiments
Wistar rats, weighing 250-300g, provided by Beijing Hua Fukang Biological Technology Co., Ltd. The experiment was divided into three groups, 8 rats in each group: normal control (NC) group, model control (MC) group, and DHZCP group. The rats in normal control group were subcutaneously injected with normal saline, the rats in model control and DHZCP group were subcutaneously injected with 40% CCl 4 solution (5 ml/kg) at the rst time, then were subcutaneously injected with 40% CCl 4 solution (3 ml/kg) every 3 days for 4 weeks to establish the hepatic brosis model [15]. At the end of the 4th week, two rats in each group were randomly executed to con rmed the formation of hepatic brosis by pathological examination. The rats in DHZCP group were given intragastric administration 3.3 g/kg DHZCP from the fth week six weeks. The rats in NC and MC groups were given intragastric administration equivalent volumes of normal saline.By the end of the experiment, no rat death occurred in each group. The experiment were approved by the Ruikang Hospital Ethical Committee.
Liver tissues were routinely excised, then washed, dehydrated, embedded in para n, and sliced. The slices were stained with hematoxylineosin (H&E). A Ishak scoring systemwas applicated to evaluate the liver brosis [16].
In order to isolate primary HSCs for western blot, the liver were perfused with 0.05% enzyme protease and 0.025% collagenase, re-digested with DNase for 45 min. Centrifugation of liver tissue was performed to remove hepatocytes and to get hepatic Non-parenchymal cells (NPC). Hepatic NPC solution was added with 70 and 30 diluted Percoll cell separation solution. After centrifugation, HSCs are located between hepatic NPC solution and 30% Percoll solution, and Kupffer cells are located between 30% Percoll solution and 70% Percoll solution. Primary HSCs were identi cated by immunocytochemical staining of α-SMA. Four elds were selected in each slice to evaluate the average optical density by Image-Pro Plus 6.0(Media Cybernetics, Rockville, USA).

Preparation of DHZCP containing serum
Wistar rats, in drug-containing serum group were intragastrically administered with 3.3 g/kg DHZCP for 7 days, and rats in normal serum group were given equivalent volumes of normal saline. Blood was collected 1h after the last Administration from the rats abdominal aorta and placed in a 10ml centrifuge tube. After the blood clot is well contracted, the serum was aseptically separated at 3000 r/min for 15 min, then the serum was inactivated at 55°C for 30 min and stored at −20°C for use.

HSC-T6 cells culture and cell grouping
The HSC-T6 cells were provided by the cell bank of the Chinese Academy of Sciences in Shanghai. HSC-T6 cells were cultured in culture media containing 10% high-quality fetal bovine serum and 1% penicillinstreptavidin. HSC-T6 cells were subcultured when con uent at a 1:3 split ratio, and cultured at 37°C, 5% CO 2 and saturated humidi ed incubator.
HSC-T6 cells were randomly divided into NC group DHZCP group LPS group, and added LPS DHZCP+LPS group. The NC group were cultured regularly for 48 h; the DHZCP group were given 20% DHZCP containing serum for 48 h; the LPS group were cultured for 44 h at rst, then were given 100 ng/ml LPS for 4 h; the DHZCP+LPS group were given 20% DHZCP containing serum for 44 h at rst, and then were given 100 ng/ml LPS for 4 h. The experiment was repeated 2 times, each group had three samples.

Western blot analysis
Primary HSCs and HSC-T6 cells were collected and subjected to electrophoresis (Bio-Rad, Hercules, USA). Then the cells were transferred to a membrane(Bio-Rad, Hercules, USA) from gel, blocked, and incubated with a diluted primary antibody 90 min, and incubated with secondary antibody 90 min. Membranes were exposured with chemiluminescence (PIERCE, Holmdel, USA). The relative density of each protein band is the ratio to GAPDH.
Immunohistochemical analysis HSC-T6 cells of all groups were prepared. Slices were treated with 3% H 2 O 2 to inhibite the endogenous peroxidase activity, then were blocked with 10% goat serum. Primary antibodies targeting TLR4 MyD88 and BAMBI (dilution 1:400) were applied. After phosphate buffered saline (PBS) poaching, the secondary antibody and peroxidase were used. The activity of peroxidase was shown with diaminobenzidine. Then the slices were stained with hematoxylin. Four elds were selected in each slice to evaluate by semiquantitative integration method [17]. The semi-quantitatively scores is the sum of the integral of the tinctorial strength and the integral of proportion of positive cells. The proportion of positive cells is < 5% for 0, 5% to 25% for 1 point, 26% to 50% for 2 points, 51% to 75% for 3 points, and > 75% for 4 points.
Integral of the tinctorial strength: 0 is for colorless, 1 point is for light yellow, 2 points is for brown, and 3 points is for tan.

Electrophoretic mobility shift assay (EMSA) analysis
The nuclear protein components extracted from HSC-T6 cells and the light shift Chemiluminescent EMSA Kit (PIERCE, Holmdel, USA) was used for gel migration assay. The probe was synthesized and puri ed by Shanghai Bioengineering Co., Ltd. The sequence of NF-κB probe were: 5'-AGT TGA GGG GAC TTT CCC AGG C -3', and 3'-TCA ACT CCC CTG AAA GGG TCC G-5'.
Statistical analysis Data analysis were performed by SPSS Version 21.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was regarded as signi cant.

Results
DHZCP alleviated the liver brosis in rats hepatic lobule structures was clear and hepatic cell cords were arranged radically around the central veins in NC group; severe macrosteatosis, numerous complete pseudo lobules and brous septa were observedin MC group ; In comparison, few pseudo lobules and brous tissue were found in DHZCP group ( Figure 1). The hepatic brosis in NC group were all in S0 phase which had a signi cant difference with MCgroup (P < 0.05). The hepatic brosis phases of DHZCP group mainly distributed in the S1-S2 phases, were better than the MC group in the S3 phase (P < 0.05, Table 1).

DHZHP inhibited primary HSCs activation in rats
The main characteristics of HSCs activation are proliferation and myo broblastic transformation. The marker of activated HSCs is α-SMA, positive staining of α-SMA was visible in each group, but much deeper positive staining of α-SMA was observed in MC group. However, weaker staining was observed in the DHZCP group compared with MC group (Figures 2). The average optical density of α-SMA in each group was obtained by image analysis and statistical analysis. The average optical density of α-SMA in NC group MC group and DHZCP group were 29.69 ± 8.23, 57.73 ± 25.48, 35.37± 16.08. The average optical density of α-SMA in MC group was higher than that of NC group (P < 0.05), the average optical density of α-SMA in DHZCP group was lower than that MC group (P < 0.05).
The effect of DHZCP on associated molecular of NF-κB activation in primary HSCs Primary HSCs were isolated from liver tissue of rats in different groups, to investigate the effect of DHZCP on associated molecular of NF-κB activation in primary HSCs, signal molecules expression were detected by Western blot and EMSA. The protein expression of TLR4 and MyD88 were up-regulated (P < 0.05, Figure 3A and 3B), and BAMBI was down-regulated in the MC group compared with NC group (P < 0.05; the TLR4 and MyD88 were down-regulated, and BAMBI was up-regulated signi cantly in the DHZCP group than the MC group (P < 0.05). The activity of NF-κB increased signi cant in MC group compared with another two groups (P was both < 0.001), but decreased in DHZCP group compared with MC group (P < 0.001, Figure 3C and 3D).
Immunohistochemical analysis were used in this part study.Compared with the DHZCP group, the expression of TLR4 and MyD88 cells were higher, but the expression of BAMBI was lower in the LPS group; compared with the LPS group, TLR4 and MyD88 was lower, BAMBI was increased ( Figure 4A). The semi-quantitative scores of TLR4 and MyD88 were increased (P was both < 0.05), BAMBIwas decreased in LPS group compared with NC group (P < 0.05); TLR4 and MyD88 were decreased and BAMBI was increased in DHZCP+LPS group compared with LPS group (P was both < 0.05, Figure 4 B).TLR4, MyD88 and BAMBI showed similar results with Immunohistochemical analysis by Western blot in HSCs-T6 cells ( Figure 5A and 5B).

DHZCP inhibited the activity of NF-κB induced by LPS in HSC-T6 cells.
The activity of NF-κB was signi cantly higher in LPS group compared to other groups (P was all < 0.001); after DHZCP containing serum intervention, NF-κB was decreasedsigni cantly (P < 0.001, Figure 6).
DHZCP is a TCM ancient formula from "Jin Kui Yao Lue", authorized by Zhang Zhongjing, The components in DHZCP had the effect of promoting blood circulation to removing blood stasis and strengthening the body resistance, which in accordance with the clinical pathogenesis of hepatic brosis [23,24]. In our previous study, we used CCl4 to induced liver brosis model and found that DHZCP could alleviate liver brosis and improve the level of serum markers of liver function(alanine aminotransferase, aspartate aminotransferase, TGF-β1 and LPS) [25]. In this study,the effects of DHZCP on the histological changes in liver also revealed that DHZCP had signi cant effects on improving the liver tissue morphology and ameliorated hepatic brosis in rats.
Some studys have showed DHZCP regulated the level of in ammatory factors, reduced the deposit of collagen I, and further inhibited the apoptosis of hepatocytes [26]; DHZCP regulated the expression of metalloproteinases1 mRNA and increased the expression of matrixmetalloproteinase1 mRNA in rats with liver brosis [27]. However, whether the DHZCP interfered with LPS-activated NF-κB signal pathway was not clear.
HSCs were very essential to the generate and development of hepatic brosis. Persistent in ammatory stimulation resulted in the transition of HSCs into a myo broblast, the expression of α-SMA is one of the main characteristics of its activation [28]. As shown in Fig. 2, the expression of α-SMA was smaller in liver slices of normal rats, but increased in model group. Administration of DHZCP resulted in decrease in α-SMA expression.
Activated HSCs expresses TLR4 and had a higher response to LPS [21]. LPS was a de nitive ligand of TLR4, it activated HSCs through the MyD88-NF-κB pathway. In our study, the expression of TLR4 and MyD88 in HSCs was increased, the expression of BAMBI was decreased in rats with hepatic brosis, and the activity of NF-κB was signi cantly highe.Further more TLR4 and MyD88 and the activity of NF-κB was inhibited, and the BAMBI was promoted after DHZCP treat.
We then performed experiment on HSC-T6 cells by use of DHZCP drug-containing serum. This method re ected the actual effect of drugs in vivo and was be used widly [29]. In our study, we found that there was no signi cant difference in the protein expression of TLR4, MyD88, NF-κB and BAMBI between the NC group and DHZCP group, suggesting that DHZCP may not directly target at HSCs. BAMBI was a TGFβ pseudoreceptor that silences TGF-β signaling [30], only one LPS-related gene signi cantly downregulated in 121 gene in HSCs regulated by LPS [3]. In the present study, LPS induced HSC-T6 cells to express TLR4, activated the in ammatory signal pathway, enhanced the activity of NF-κB, and decreased the expression of BAMBI. While LPS-induced HSC-T6 cells were treated with DHZCP containing serum, the protein expression of TLR4 and MyD88 decreased, the activity of NF-κB protein decreased, and the expression of BAMBI increased.

Conclusion
Chronic hepatitis eventually develops into liver brosis, cirrhosis, and even liver cancer. Therefore, it is very important to intervene the pathological procession of liver brosis. With multilevel, multichannel, multitarget pharmacological effects, TCM has been selected as an alternative treatment for liver brosis. DHZCP has been well recorded therapeutic e cacies since thousands of years in the past, and has been applied in the clinical treatment of liver brosis. This study provides a new point to clarify the mechanism for the application of DHZCP in liver brosis. DHZCP inhibited NF-κB activation induced by LPS, and upregulated BAMBI expression in HSCs. This was one of important mechanisms of DHZCP in improving brosis.

Declarations
Ethics approval and consent to participate The experimental protocol was established according to the ethical guidelines and was approved by the author's Ethical Committee.

Consent for publication
The manuscript is approved by all authors for publication.
Availability of data and materials Table   Table 1 The phase of hepatic fibrosis   DHZCP alleviated the liver brosis in rats. H&E staining was performed to detect morphological structure of each group (× 100).  Effects of DHZCP on α-SMA expression in each group, protein expression of α-SMA in primary HSCs were detected by immunohistology (×100).

Figure 2
Effects of DHZCP on α-SMA expression in each group, protein expression of α-SMA in primary HSCs were detected by immunohistology (×100).

Figure 2
Effects of DHZCP on α-SMA expression in each group, protein expression of α-SMA in primary HSCs were detected by immunohistology (×100).