Citation: Jiang SL, Hu XD, Liu P. Immunomodulation and liver protection of Yinchenhao decoction against concanavalin A-induced chronic liver Injury in mice. J Integr Med. 2015; 13(4): 262–268.
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Autoimmune liver disease is a chronic progressive inflammatory liver disease in which the immune system attacks the normal liver cells and causes liver damage. It more commonly occurs in females. Treatment of autoimmune liver disease is based on the long-term use of corticosteroids with or without the additional use of immune system suppressors, such as azathioprine, to inhibit abnormal immune response. Although these medications may show improvements in liver functions and prevent further autoimmune-mediated liver damage, they usually have severe side effects, including osteoporosis, diabetes, high blood pressure, immune tissue degeneration and hair and skin problem; these adverse effects may be worse in patients that have developed cirrhosis. Therefore, identifying safe and effective new ways to treat autoimmune liver disease remains of important clinical relevance.
Yinchenhao decoction is a famous traditional Chinese medicine prescription initially described in the book entitled Treatise on Febrile Diseases (Shanghan Lun), written by Zhongjing Zhang, during the Han Dynasty. It comprises Yinchenhao (Herba Artemisiae Capillaris), Zhizi (Fructus Gardeniae) and Dahuang (Radix et Rhizoma Rhei Palmati). It is one of several traditional Chinese medicinal prescriptions that are widely used for treating various liver diseases such as acute or chronic hepatitis, fatty liver and cirrhosis. Experimental studies have shown that Yinchenhao decoction can protect liver against concanavalin A (ConA)-induced acute inflammation and liver injury in mice[3,4]. However, it remains unclear whether Yinchenhao decoction may protect the liver against chronic immune/inflammatory response-mediated liver damage. In this study, we have applied a chronic liver injury mouse model, induced by repeated injection of ConA, to investigate the immune regulatory and liver protective effects of Yinchenhao decoction, and compare with the effects of dexamethasone.
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2 Materials and methods
Yinchenhao decoction was prepared by the Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, China. Briefly, Yinchenhao 60 g, Zhizi 30 g, and Dahuang 20 g were mixed, and extracted twice with water (500 mL × 2) at 100 ℃ for 1 h each time. The supernatants from each extraction were combined for a total volume of 250 mL. The final concentration of the crude drug was 40 mg/mL. Dexamethasone (Catalog No. D4902) and ConA (Catalog No. C2010) were purchased from Sigma (St. Louis, MO, USA).
Female BALB/c mice (B&K Universal Group Ltd., Shanghai, China) were kept on a 12-hour light/dark cycle in the Animal Lab Center of the Shanghai University of Traditional Chinese Medicine, with free access to mouse chow and water. The mice, body weight (18 ± 2) g, were divided into 4 groups at random: normal control (n = 10), ConA model (n =14), ConA model treated with Yinchenhao decoction (n = 14) and ConA model treated with dexamethasone (n = 14). Yinchenhao decoction (400 mg/kg body weight) and dexamethasone (0.5 mg/kg body weight, dissolved in saline) were administered orally by gavage at a dose of 0.01 mL/g body weight, once a day for 28 d. The doses of Yinchenhao decoction and dexamethasone were chosen according to previous reports that have shown their protective roles in ConA-induced acute liver injury mouse models[4,5]. Normal saline (0.01 mL/g body weight) was given by oral gavage to mice in the normal control and ConA model groups. All ConA model mice received tail vein injections of ConA (10 mg/kg) on days 7, 14, 21 and 28, at 1 h after treatments with Yinchenhao decoction, dexamethasone or normal saline. The mice were euthanized for sampling at 8 h after the last ConA injection. The mice were bled for serum isolation used for liver function assays by determining serum alanine aminotranferease (ALT) activity and albumin level using commercial kits (Nanjing Jiancheng Biological Engineering Institute, China). Liver and spleen samples were harvested and weighed. For histology, a piece of liver tissue (about 1.0 cm × 0.8 cm × 0.3 cm) from each mouse was fixed with 10% buffered formalin acetate, processed (dehydration with 70%, 90%, and 100% ethanol, followed by clearing with xylene), and embedded in paraffin. Total RNA was extracted from liver tissues following the manufacturer’s protocol using Aqua-Spin RNA Isolation Mini Kit (Shanghai Huashun Biotechnology Co., Ltd, China). The study protocol was approved by the Shanghai University of Traditional Chinese Medicine’s Animal Ethics Committee (Shanghai, China).
2.3 Histological analysis
Liver tissue sections (5 μm) were stained with hematoxylin and eosin (H&E). Hepatic inflammation activities were evaluated according to Tailing Wang’s revised Knodell histologic activity index for scoring chronic hepatitis[6,7], which includes portal inflammation (P), intralobular degeneration and focal necrosis (L), periportal necrosis (PN) and bridging necrosis (BN). A score was assigned to each parameter as follows: (0) none, (1) mild, (3) moderate and (4) marked. The overall numerical score was calculated by the formula: P + L + 2 (PN + BN).
2.4 Real-time polymerase chain reaction
First strand cDNA was synthesized from 1 μg of total RNA using avian myeloblastosis virus (AMV) reverse transcriptase and random 9 mers (Fermentas). RNA expression levels were determined by real-time polymerase chain reaction (PCR) using SYBR Green premix Ex Taq (Takara) and target gene-specific primers (Table 1).
2.5 Statistical analysis
Data are expressed as mean ± standard deviation. One-way analysis of variance followed by Newman-Keuls multiple comparison test was performed for statistical analysis with the threshold for significant comparisons being set at P < 0.05.
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3.1 Survival rate, body weight, liver weight, and spleen weight
As shown in Table 2, the ConA model mice had a survival rate of 78.57%; during the 28-day experiment period two ConA mice died on day 8 and 1 died on day 15. Treatment of the ConA model mice with Yinchenhao decoction enhanced the survival rate to 92.85% with 1 mouse dying on day 8. No deaths occurred in the normal control group or the ConA model mice treated with dexamethasone.
The average body weight in the ConA model group was greater relative to the normal control group (P < 0.01). However the liver and spleen weight relative to the body weight showed no significant difference between the two groups. There was no significant difference in the ratio of liver weight/body weight among the four groups. Treatment of ConA model mice with Yinchenhao decoction reduced the spleen weight (P < 0.01), but did not significantly change the ratio of spleen weight/body weight, in comparison to the ConA model mice without treatment. Notably, dexamethasone treatment significantly reduced the mouse body weight (P < 0.01), and dramatically reduced spleen weight (P < 0.01), leading to a significant reduction in the ratio of spleen weight/body weight (P<0.01), in comparison to either the normal control or the ConA model mice (Table 3).
3.2 Yinchenhao decoction reverses ConA-induced changes in serum ALT activity and Alb level
Mice with ConA-induced liver damage had significantly increased serum ALT activity (P<0.01) and reduced serum Alb level (P<0.05) relative to the normal control (Table 4). Yinchenhao decoction treatment completely reversed these markers of ConA-induced damages in liver function. Treatment with dexamethasone also prevented the decrease in serum Alb level in the ConA model mice, but did not prevent the increase in serum ALT activity.
3.3 Yinchenhao decoction attenuates ConA-induced liver tissue pathological changes
H&E staining of the liver showed that mice receiving ConA alone had increased hepatocyte swelling, degeneration, and piecemeal or focal necrosis in intralobular, periportal, and bridging areas. Mice receiving ConA alone also showed marked inflammatory infiltration (mostly lymphocytes) that was obvious in particular portal areas (Figure 1B). Treatment with Yinchenhao decoction clearly attenuated the extent of the liver pathological changes induced by ConA. There was no hepatocyte degeneration found in the liver sections from Yinchenhao decoction-treated mice (Figure 1C). Treatment with dexamethoasone also eliminated necrosis and inflammatory cell infiltration, however, in liver sections from these mice, hepatocyte degeneration was observed extensively (Figure 1D). The overall Knodell histologic activity index was significantly higher in ConA model mice than in the normal control mice, and was improved in the mice treated with either Yinchenhao decoction or dexamethasone (Table 5).
3.4 Yinchenhao decoction modulates ConA-induced immunoregulatory gene expression in liver tissues
On day 28 of the experiment, expression of IFN-γ, IL-4, MCP-1, and CD163 was significantly increased in ConA mice relative to normal control (P<0.01, P<0.05). At the same time ConA mice had significantly lower levels of TNF-α and IL-6 expression relative to normal control (P<0.01 and P<0.05) (Table 6). These changes are representative of a chronic inflammatory response. Treatment with Yinchenhao decoction completely reversed the changes in IFN-γ, IL-4, MCP-1, CD163, TNF-α and IL-6 gene expression caused by ConA. Although treatment with dexamethasone reversed ConA-induced changes in IL-4, MCP-1, TNF-α, and IL-6 mRNA levels, it had no effect on the enhanced mRNA expression of IFN-γ and CD163 caused by ConA.
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In the present study, we have demonstrated the protective role of Yinchenhao decoction in a repeated ConA injection-induced chronic liver injury mouse model. Treatment with Yinchenhao was able to reduce serum ALT activity, increase serum Alb concentration and reduce or eliminate liver inflammation and necrosis. Although dexamethasone treatment prevented ConA-induced changes in serum ALT activity and Alb concentration and inhibited liver inflammation, it caused extensive hepatocyte degeneration and significantly reduced mouse body weight and spleen weight. Treatment with Yinchenhao decoction did not have any of these side effects.
ConA is a lectin originally extracted from the jack-bean, Canavalia ensiformis. In 1992, Tiegs et al reported for the first time that a single tail vein injection of ConA in mice can activate T lymphocytes and liver Kupffer cells, and induce cytokine-dependent acute liver injury. However, this acute liver injury model is not suitable to investigate the pathological mechanism of chronic liver injury because the inflammatory/immune responses are quite different from the chronic liver injury condition. In 1999, Kimura et al developed a chronic liver injury mouse model that was induced by repeated ConA injections. This chronic liver injury model shows features similar to that of autoimmune liver disease with T cell activation and chronic inflammation. Therefore, it has been considered to be a model that mimics human autoimmune liver disease and is suitable to be used for the screening and investigation of candidate drugs for the treatment of chronic autoimmune liver injury. In the present study, the liver gene expression pattern in the mice receiving repeated ConA injections showed a chronic inflammatory/immune response, featuring significantly increased expression of IFN-γ, IL-4, MCP-1 and CD163 mRNA levels as well as significantly decreased expression of mRNA for TNF-α and IL-6 , which are usually related to acute inflammatory response.
Liu et al reported that Yinchenhao decoction attenuated the liver pathological changes in dimethylnitrosamine-induced liver fibrosis in rats. Yamashiki et al previously reported that pretreatment of the mice with Yinchenhao decoction for one week protected the liver from acute injury in mice induced by a single tail vein injection of ConA; these animals showed attenuated ConA-induced increase in serum aminotransferase and lactate dehydrogenase activities, reduced inflammatory infiltration and necrosis in liver tissues, associated with decreased serum IFN-γ, IL-12 and IL-2 levels as well as increased IL-10 level. Cai et al observed a similar protective role of Yinchenhao decoction in a ConA-induced acute liver injury mouse model and found that Yinchenhao decoction reduced TNF-α expression through inhibition of NF-κB activation. These studies suggest that Yinchenhao decoction can attenuate acute liver injury through modulating the expression of inflammatory cytokines.
Our results indicate that Yinchenhao decoction had a protective role in chronic liver injury mouse model induced by repeated tail vein injection of ConA, consistent with its protective role observed in previous acute liver injury models. We further demonstrated that the protective role of Yinchenhao decoction in the chronic liver injury model was associated with a reduction of ConA-induced expression of IFN-γ, IL-4, MCP-1 and CD163. MCP-1 is a chemokine that exhibits a chemotactic activity for monocytes, memory T lymphocytes and antigen-presenting cells[10,11]. Over expression of MCP-1 in liver tissues is implicated in the pathogenesis and progression of autoimmune liver disease. Yinchenhao decoction reduced the liver expression of MCP-1, which could be one of the mechanisms by which Yinchenhao decoction attenuates inflammatory infiltration and protects the liver against T cell-mediated chronic autoimmune injury. Although overexpression of TNF-α and IL-6 in the liver is critical for the induction of the acute-phase response and injury, low levels of TNF-α and IL-6 expression could be protective[12–15]. Our results showed that TNF-α and IL-6 expression in the liver was down-regulated in the ConA-induced chronic liver injury mouse model. This alteration could result from the enhanced expression of IL-4, which could inhibit TNF-α and IL-6 expression. Yinchenhao decoction and dexamethasone prevented the ConA-induced increase in IL-4 expression and returned TNF-α and IL-6 mRNA expression to the normal levels. It has been reported that IL-6 protects the liver against T-cell mediated injury[13–15] and promotes hepatocyte proliferation. The significant reduction of serum Alb concentration in the chronic liver injury mouse model induced by repeated ConA injection may not only result from injury-caused liver dysfunction, but also relate to the inhibition of Alb synthesis by the increased IL-4 levels. The role of Yinchenhao decoction in reducing liver expression of IL-4, therefore, may also contribute to its role in reversing serum Alb levels. CD163 is a scavenger receptor and cell surface marker of monocyte/macrophage?lineage including Kupffer cells. The enhanced expression of CD163 in the liver tissues of mice with repeated ConA injection was consistent with the increased inflammatory cell infiltration, and both were reversed by treatment with Yinchenhao decoction.
Taken together, our results indicate that Yinchenhao decoction can modulate expression of multiple genes related to inflammatory/immune response, attenuate liver injury and improve liver function in a chronic autoimmune liver injury mouse model induced by repeated injection of ConA, and that Yinchenhao decoction showed no effect in causing hepatocyte degeneration and spleen weight loss, a side effect that could be seen in long-term treatment with dexamethasone.
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5 Funding and acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 90409020) and the College Young Teacher Training Program of Shanghai Municipal Education Commission (No. ZZszy13022).
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6 Conflict of interests
The authors declare that they have no conflict of interests.
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Figures and Tables in this article:
Figure 1 Representative images showing H&E staining of mouse liver tissue sections (×200)
A: Normal control; B: ConA + saline; C: ConA + Yinchenhao decoction; D: ConA + dexamethasone. H&E: hematoxylin and eosin.
Table 1 Primers used in real-time polymerase chain reaction
IFN-γ: interferon-γ; IL-4: interleukin-4; TNF-α: tumor necrosis factor-α; MCP-1: monocyte chemotactic protein-1; CD163: cluster of differentiation 163; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
Table 2 Survival rate in mice with different treatments
Table 3 Body weight, liver weight, and spleen weight in mice with different treatments
Data were represented as mean ± standard deviation. **P<0.01, vs normal control; △△P<0.01, vs Con-A + saline.
Table 4 Serum ALT activity and Alb level in mice with different treatments
Data were represented as mean ± standard deviation. *P<0.05, **P<0.01, vs normal control; △△P<0.01, vs Con-A + saline. ALT: alanine aminotransferase; Alb: albumin.
Table 5 Overall Knodell histologic activity index for scoring chronic hepatitis
Data were represented as mean ± standard deviation. **P<0.01, vs normal control; △△P<0.01, vs Con-A + saline.
Table 6 Comparison of immunoregulatory gene mRNA expression levels in liver tissues in mice with different treatments
The mRNA results were normalized to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and expressed as fold-changes compared to normal control levels that were set as 1-fold. Data are represented as mean ± standard deviation. *P<0.05, **P<0.01, vs normal control; △P<0.05, △△P<0.01, vs Con-A + saline. IFN-γ: interferon-γ; IL-4: interleukin-4; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; MCP-1: monocyte chemotactic protein-1.
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