Hepatoprotective effect of capsaicin against concanavalin A-induced hepatic injury via inhibiting oxidative stress and inflammation

Abstract

Immune-mediated liver injury plays a crucial role in the pathogenesis of liver diseases, which can result from viral infections, autoimmunity, alcohol intake, and drug use. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model with similar pathophysiology to that of human viral and autoimmune hepatitis. Capsaicin, a selective agonist of the transient potential vanilloid subfamily member 1 (TRPV1) receptor, exhibits anti-inflammatory effects on various causes of inflammation. In the present study, we investigated the effect of capsaicin on Con A-induced hepatitis. Capsaicin (1 mg/kg body weight) was administered by intraperitoneal injection, after which (30 minutes), the mice were challenged intravenously with Con A (20 μg/g body weight). We collected serum for plasma transaminase analysis. Pro-inflammatory cytokine levels and hepatocyte apoptosis were assayed by ELISA and TUNEL, respectively. Liver samples were collected for real-time PCR, hematoxylin and eosin staining, and measuring oxidative stress and myeloperoxidase levels. Activation of splenocytes and hepatic mononuclear cells was analyzed by flow cytometry. Compared with control, the capsaicin-treated group showed significantly decreased aminotransferase levels and markedly prolonged mouse survival. Capsaicin pretreatment also attenuated hepatocyte apoptosis and oxidative stress. Furthermore, tumor necrosis factor-α and interferon-γ levels in serum and liver were significantly suppressed, while the percentage of myeloid-derived suppressor cells increased after capsaicin pretreatment. Our findings indicate that capsaicin pretreatment protects mice from Con A-induced hepatic damage and is partially involved in inhibiting hepatocyte apoptosis, oxidative stress, and inflammatory mediators as well as regulating activation and recruitment of intrahepatic leukocytes.

Keywords: Concanavalin A; capsaicin; hepatitis; inflammation; oxidative stress.

Figure 1

Capsaicin (CAP) pretreatment alleviated concanavalin A (Con A)-induced hepatic injury. Mice were treated with PBS or capsaicin 30 min before being challenged with Con A (20 μg/g body weight). Twelve hours after Con A injection, serum and livers were collected. A. Alanine aminotransaminase (ALT) levels in serum. The data represent the means ± standard deviation (SD; n = 6-8). **P < 0.01. B. Survival experiments were performed with mice treated with a lethal dose of Con A (25 μg/g body weight; n = 8-10). C. Liver sections (Con A or CAP/Con A group) were stained with hematoxylin and eosin (H&E). Original magnification, 100 × and 200 ×.

Figure 2

Capsaicin prevents hepatocyte apoptosis in Con A-induced hepatitis. Mice were treated with PBS or capsaicin, after which Con A was injected (20 μg/g body weight) 30 min later. A. Liver tissues were collected 12 h after Con A administration for TUNEL staining (original magnification, 100 × and 200 ×). B. Bcl-2 and Bax mRNA levels in the liver were assessed by real-time PCR. The data represent the means ± SD (n = 6-8). *P < 0.05; **P < 0.01.

Figure 3

Effects of capsaicin on oxidative stress and myeloperoxidase (MPO) levels in liver tissue. Mice were treated with PBS or capsaicin 30 min before being challenged with Con A. Liver tissues were collected at the indicated time points after Con A injection. (A) Superoxide dismutase (SOD), (B) malondialdehyde (MDA), and (C) MPO levels in the liver were tested. The data represent the mean ± SD (n = 6-8). *P < 0.05; **P < 0.01.

Figure 4

Capsaicin pretreatment inhibited cytokine release in Con A-treated mice. Mice were treated as described in Figure 3. Serum and liver tissue were collected at the indicated time points after Con A injection. A. TNF-α and IFN-γ levels in serum were measured by ELISA. The data represent the means ± SD (n = 6-8). *P < 0.05; **P < 0.01. B. mRNA levels of IFN-γ and TNF-α in liver tissues were determined by real-time PCR. The data represent the means ± SD (n = 6-8). *P < 0.05; **P < 0.01.

Figure 5

Capsaicin pretreatment inhibited T lymphocyte activation in the liver. Mice were treated as described in Figure 3. Hepatic mononuclear cells (MNCs) were prepared 12 h after Con A injection and were analyzed by FACS using PE-conjugated anti-CD3, FITC-conjugated anti-CD69, and APC-conjugated anti-NK1.1 antibodies. A, B. The effects of capsaicin on the percentage of CD69⁺ T cells (CD3⁺), NK cells (CD3^- NK1.1⁺), and NKT cells (CD3⁺ NK1.1⁺). C, D. Percentages of T cells (CD3⁺), NK cells (CD3^- NK1.1⁺), and NKT cells (CD3⁺ NK1.1⁺). Data represent the means ± SD (n = 5-7). *P < 0.05; **P < 0.01.

Figure 6

Capsaicin pretreatment increased the percentage of myeloid-derived suppressor cells (MDSCs) in the liver. Hepatic MNCs were isolated as described in Figure 5 and were analyzed by FACS with FITC-conjugated anti-CD11b Ab, APC-conjugated anti-Gr-1 Ab, PE-conjugated anti-Ly6G Ab, and APC-conjugated anti-Ly6C antibodies. The percentages of (A, C) CD11b⁺Gr-1⁺ MDSCs as well as (B, D) CD11b⁺Ly6C^intLy6G^high granulocytic and CD11b⁺Ly6C^highLy6G^low monocytic MDSCs. Data represent the means ± SD (n = 5-7). *P < 0.05; **P < 0.01.