A polysaccharide from Dendrobium huoshanense prevents hepatic inflammatory response caused by carbon tetrachloride

Dendrobium huoshanense is a precious herbal medicine in China, which exhibits a variety of restorative and therapeutic effects. This study aimed at investigating the hepatoprotective effects of a polysaccharide (DHP1A) isolated from D. huoshanense via water extraction, diethylaminoethyl (DEAE) cellulose anion exchange and size exclusion chromatography. The animal experiment indicated that the oral administration of DHP1A obviously reduced the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and 8-hydroxy-2′-deoxyguanosine in the serum of mice treated with carbon tetrachloride (CCl4), suggesting the hepatoprotective potential of this polysaccharide. Moreover, DHP1A decreased the expressions of tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein-1, macrophage inflammatory protein-2, CD68 and phosphorylated IκBα (p-IκBα) in the CCl4-treated mice. These results revealed that the hepatoprotective effect of DHP1A was partly attributed to its anti-inflammatory action.


Introduction
Dendrobium huoshanense, a perennial orchid species, has been used as a folk medicine in China for centuries. Its stems possess variety of restorative and therapeutic functions, which has been exploited as a valuable beverage, named as 'ShiHu'. Pharmacological research works indicate that polysaccharides are the major active components in D. huoshanense. Through in vitro assays, it was reported that the polysaccharides from D. huoshanense were capable of activating immune cells, [1] scavenging free radicals [2] and inhibiting glycation. [3] Moreover, the in vivo assays have demonstrated that oral administration of D. huoshanense polysaccharides can attenuate the streptozotocin-induced cataract [4] and modulate the immune responses in intestines, spleen and liver. [5] Carbon tetrachloride (CCl 4 ), a potent hepatotoxic chemical, can rapidly cause an oxidative stress and inflammatory response to hepatocytes, which leads to an acute hepatic necrosis. Excessive production of free radicals (including ¢CCl 3 and ¢OOCCl 3 ) originating from the cytochrome P450 metabolism of CCl 4 is responsible for the initiation of liver injury, and the consequent inflammatory response that further aggravates the extent of injury. [6] Some studies suggest that the activation of intrahepatic macrophages (i.e. Kupffer cells) plays a key role in the CCl 4 -induced inflammatory response via release of various cytokines and chemokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, IL-6, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein-2 (MIP-2). [7,8] Besides, the nuclear factor-kappa B (NF-kB) is believed to be associated with the hepatic inflammatory injury induced by CCl 4 . [9] In recent years, natural polysaccharides have been reported to be capable of enhancing liver resistance against CCl 4 toxicity.[10À12] Huang et al. [13] previously reported that the crude polysaccharides from D. huoshanense could protect the liver from CCl 4 hepatotoxicity, but the constituents in the crude D. huoshanense polysaccharides were so complex that the relationship between the hepatoprotection and polysaccharides still remained obscure. Moreover, many views considered that the protective effects of polysaccharides on CCl 4 -induced liver injury were mainly due to the antioxidative properties of the polysaccharides, but investigations concerning the mechanisms of inflammatory regulation facilitated by polysaccharides were relatively limited. In a previous work, a homogenous polysaccharide (named as DHP1A) was obtained from D. huoshanense via a water extrac-tionÀethanol precipitation, anion exchange and size exclusion chromatography, and its structural characterization was performed by the various spectral methods. [14] In this study, we further assessed the protective effects of DHP1A on CCl 4 -induced liver injury, and proposed that the inhibition of inflammatory responses may be *Corresponding author. Email: jianpingluo@hfut.edu.cn an important hepatoprotective mechanism of this polysaccharide.

Animals
Male Kunming mice (SPF grade, 23 § 2 g) were obtained from the Experimental Animal Center of Anhui Medical University, China. The mice were housed in an air-conditioned room (25 § 2 C) with a normal light/night cycle. The animal care and experimental protocols complied well with the national guidelines for the care and use of animals.

Preparation of DHP1A
The preparation of DHP1A from crude polysaccharides of D. huoshanense was carried out according to previous methods. [14] DHP1A is proved to be a homogenous polysaccharide fraction with a molecular weight of 6700 Da and consists of mannose, glucose and galactose in the molar ratio of 2.5:16.0:1.0, which was evidenced by high performance liquid chromatography, gas chromatography-mass spectrometer and nuclear magnetic resonance. The carbohydrate concentration of DHP1A was determined to be 98.9% using phenolÀsulphuric acid method, [16] while proteins were not detected.

Experimental design
After acclimatized for 7 days, 80 mice were randomly divided into eight groups (n D 10 per group), including (1) blank control group (distilled water); (2) silymarin treatment group (25 mg/kg body weight (BW)); (3) lowdose DHP1A treatment group (100 mg/kg BW); (4) highdose DHP1A treatment group (200 mg/kg BW); (5) CCl 4 group (0.2% CCl 4 dissolved in olive oil, 10 mL/kg BW); (6) silymarin (25 mg/kg BW) C CCl 4 treatment group; (7) DHP1A (100 mg/kg BW) C CCl 4 treatment group and (8) DHP1A (200 mg/kg BW) C CCl 4 treatment group. DHP1A or silymarin was dissolved in distilled water and orally administered to the mice at the dose of 10 mL/kg BW once daily for 14 days. The control group (1) and CCl 4 group (5) were orally administered with distilled water at the same dose. After 4 hours of the last treatment, the mice (groups 5À8) were intraperitoneally injected with 0.2% CCl 4 solution at the dose of 10 mL/kg BW, while the rest mice (groups 1À4) were treated with olive oil at the same dose. Twenty-four hours later, the blood was collected from the inner canthus cave and centrifuged at 3000 rpm for 10 min to separate the serum, which was stored at ¡70 C until analysis. Subsequently, the mice were sacrificed by cervical dislocation and their livers were quickly removed. The left lobe of the liver was fixed by immersing in 10% formalin solution, while the rest of the liver was quickly frozen with liquid nitrogen and stored at ¡70 C until analysis.

Assessment of liver function
In order to evaluate the degree of liver injury, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and 8-OHdG in the serum were measured by commercially available kits.

Histopathological analysis
The hepatic tissue fixed with formalin solution was embedded in paraffin, sliced into sections of 5 mm thickness and stained with hematoxylin and eosin (H&E). The pathological changes of the liver tissues were examined using an optical microscope (Nikon, Japan) at standard magnification.

Cytokine assays
The levels of TNF-a, IL-b and IL-10 in the serum were measured by ELISA kits according to the manufacturer's instruction.

Real-time quantitative polymerase chain reaction
Total RNA was extracted from the hepatic tissue by using TRIzol reagent and reverse transcribed into first cDNA strand with iScript TM cDNA Synthesis Kit.

Immunohistochemical assay
The expression of CD68 in the livers was investigated by the immunohistochemical staining according to the manufacturer's procedure. In brief, liver tissue sections (4 mm thick) after deparaffinized and heated in boiling sodium citrate buffer solution (0.01 M, pH 6.0) for 5 min were blocked with 3% H 2 O 2 and 10% goat serum, successively and then incubated with a polyclonal rabbit anti-CD68 antibody (1:100 dilution) at 4 C overnight. After washed thrice with phosphate buffer solution (PBS), the sections were incubated with goat anti-rabbit IgG (37 C, 30 min) and S-A/HRP (37 C, 30 min), successively. At last, the sections were stained with DAB solution and hematoxylin, and viewed using a Nikon 80i microscope (Tokyo, Japan).

Western blot analysis
The western blot technique was used to analyse the expression of p-IkBa in the cytoplasm of hepatocytes according to previous protocols. [17] In short, proteins extracted from the liver tissue were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. After blocked with 5.0% skim milk, the membrane was incubated with p-IkBa primary antibody overnight at 4 C, horseradish peroxidaseconjugated secondary anti-body and chemiluminescence reagents successively. After that, the membrane was investigated by Gel Doc TM XR C and ChemiDOC TM XRS C gel documentation systems (BIO-RAD, Hercules, USA). As an internal reference, a-tubulin was used in this assay.

Statistical analysis
Data were expressed as mean § standard deviation. The statistical difference between groups was determined by one-way analysis of variance followed by the Tukey's test. Difference was considered significant when p < 0.05.

Result and discussion
It is well known that natural polysaccharides are a type of active biomacromolecules that can modulate the innate immunity and adaptive immunity of body through reacting with diverse immune cells. [18,19] Some of them have exhibited a variety of beneficial preventive and therapeutic properties, which attract more and more attention. [20] In this study, we demonstrated that DHP1A, a polysaccharide from D. huoshanense, could effectively enhance the liver resistance against CCl 4 -induced inflammatory injury.
CCl 4 -induced liver injury is a good model to evaluate hepatoprotective agents. Following the CCl 4 injection, the levels of ALT, AST and LDH in the serum of mice were increased to 2286%, 298% and 261% of the control, respectively. Moreover, the production of 8-OHdG, an oxidative product of DNA, [21] was increased to 144% of the control. These results showed that CCl 4 toxicity not only affected cell membranes but also cell nuclei. DHP1A pretreatment attenuated the increase of these biochemical parameters caused by CCl 4 , suggesting the obvious hepatoprotective effect of this polysaccharide. When the mice were pretreated with DHP1A at 200 mg/kg BW, the levels of ALT, AST, LDH and 8-OHdG following the CCl 4 treatment were 1551%, 155%, 178% and 118% of the corresponding control, respectively, which were significantly lower than these in the mice treated with CCl 4 alone ( Figure 1). Furthermore, the DHP1A treatment alone was proven to be of low toxicity. Silymarin, a hepatoprotective drug, [22] was used as a positive control, which exhibited similar results.
The results were further confirmed by the investigations of pathological liver tissue sections. As shown at Figure 2, the pretreatment of DHP1A (Figure 2(G) and 2 (H)) significantly reduced the centrilobular necrosis induced by CCl 4 as compared with the mice treated with CCl 4 alone (Figure 2(E)). Moreover, it was easy to find little difference between the normal mice and the mice pretreated with DHP1A or silymarin alone, indicating the low toxicity of DHP1A and silymarin (Figure 2(A)À(D)).
Most of liver diseases are accompanied by the inflammatory responses that stem from a systemic defence action against various tissue damages, but failure to appropriately regulate the self-response would result in serious pathological consequences. [23] In the early phase of CCl 4 hepatotoxicity, TNF-a and IL-1b have been regarded as the primary pro-inflammatory cytokines originating from activated Kupffer cells to initiate the inflammatory cascade. [10,24] As shown in Figure 3, the levels of TNF-a and IL-1b in the mice treated with CCl 4 alone were increased to 152% and 177% of the corresponding control, respectively. However, DHP1A pretreatment alleviated the changes of the two pro-inflammatory cytokines. IL-10 as an anti-inflammatory cytokine can disturb the synthesis of pro-inflammatory cytokines. [11] Following the DHP1A treatment, the IL-10 production significantly increased in the mice treated or non-treated with CCl 4 , possibly suggesting that IL-10 played a key role in modulating the inflammatory response caused by DHP1A.  TNF-a is a pleiotropic cytokine that regulate the expressions of certain chemokines and induce the recruitment of leukocytes into damaged sites. [25] MCP-1 and MIP-2 have been reported to be typical pro-inflammatory chemokines for the recruitments of neutrophil and mononuclear leukocytes, and exert important impact on the inflammatory injuries of livers. [26,27] CD68 is a glycoprotein expressed in mononuclear phagocyte lineage cells, including monocytes, Kupffer cells and most macrophages. [28] Following the CCl 4 treatment alone, the mRNA expression of MCP-1 increased to 1170% of that in the normal mice, while the mRNA expression of MIP-2 increased to 500% of that in the normal mice ( Figure 4). Meanwhile, the increased CD68 positive cells in liver tissues were obviously observed in the mice treated by CCl 4 alone, indicating the occurrence of inflammatory response ( Figure 5(E)). As compared with the mice in the model group, the mice pretreated with DHP1A exhibited a significant resistance against the expressions of these inflammatory mediators.
Besides, increasing evidence shows that the activation of NF-kB is an important mechanism involved in the CCl 4 -induced hepatotoxicity. [2,24] In normal state, NF-kB is combined with its inhibitory protein (IkB) and exists in the cytoplasm without activity. Once cells are subjected to pathological stimuli, NF-kB shifts to the nucleus following the phosphorylation and degradation of IkB, and regulates inflammatory gene expression. [9] Our results indicated that DHP1A could attenuate the phosphorylation of IkB caused by CCl 4 (Figure 6), which possibly inhibited the activation of NF-kB pathway.

Conclusions
In summary, DHP1A exhibited obvious abilities to inhibit the inflammatory response caused by CCl 4 via decreasing the expressions of inflammatory cytokines, chemokines, CD68 and p-IkBa, suggesting the hepatoprotective potential of this polysaccharide. Moreover, the low hepatoxicity of DHP1A would provide the foundation for itself as a safe food supplement for the prevention of livers disease development.