Abstract

Objectives

Limb ischemia–reperfusion (I/R) causes remote organ injury (e.g., liver injury). Oxidation and inflammation are crucial mechanisms. We investigated the effects of cepharanthine, a potent antioxidative and anti-inflammatory drug, on alleviating liver injury induced by limb I/R.

Methods

Twenty-four adult male Sprague-Dawley rats were randomized to receive sham operation (Sham), Sham plus cepharanthine, I/R, or I/R plus cepharanthine and designated as the Sham, Sham+Cep, I/R, or I/R+Cep group, respectively (n = 6 in each group). I/R was induced by applying rubber band tourniquets high around each hind limb for 3 hours followed by reperfusion for 24 hours.

Results

The plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of the Sham and Sham+Cep groups were low, and the levels of AST and ALT of the I/R group were significantly higher than those of the Sham group (both p < 0.001). By contrast, the AST and ALT of the I/R+Cep group were significantly lower than those of the I/R group (both p < 0.001). The hepatic levels of nitric oxide (NO), malondialdehyde (MDA), macrophage inflammatory protein 2 (MIP-2), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2)/prostaglandin E₂(PGE₂) of the Sham and Sham+Cep groups were also low. As expected, the NO, MDA, MIP-2, IL-6, and COX-2/PGE₂of the I/R group were significantly higher than those of the Sham group (all p < 0.001). By contrast, the NO, MDA, MIP-2, IL-6, and COX-2/PGE₂ of the I/R+Cep group were significantly lower than those of the I/R group (all p < 0.05).

Conclusion

Cepharanthine alleviates liver injury in a rodent model of limb I/R. The mechanisms may involve reducing oxidation and inflammation.

Keywords

chemokineCOX-2cytokineinflammationoxidationprostaglandin;

1. Introduction

Vital organs (e.g., the liver) are susceptible to the influence of limb ischemia and subsequent reperfusion (I/R).1, 2, 3, 4, 5, 6 The underlying mechanisms may involve oxidation and inflammation.3, 4, 5, 6 Experimental data have demonstrated that antioxidation and/or anti-inflammation therapies may alleviate the remote organ injury induced by limb I/R.3, 4, 5

Cepharanthine is an alkaloid extract from Stephania cepharantha Hayata.⁷ Cepharanthine possesses therapeutic potential and has been used in treatments of clinical conditions, including idiopathic thrombocytopenic purpura, refractory anemia, radiation-induced leukopenia, alopecia areata, and sarcoidosis.8, 9 Cepharanthine possesses potent antioxidative and anti-inflammatory capacity.8, 9, 10 In line with this notion, we speculate that cepharanthine may exert therapeutic effects and alleviate liver injury induced by limb I/R.

To elucidate further, we conducted this study. Our hypothesis was that cepharanthine can mitigate liver injury induced by limb I/R in rats. This study employed a rodent model of bilateral hind limb I/R to facilitate investigations. Assays of histology and liver function as well as oxidation and inflammation were preformed to confirm the therapeutic effects of cepharanthine and the possible underlying mechanisms.

2. Methods

This study employed a total of 24 adult male Sprague-Dawley rats (200–250 g; BioLASCO Taiwan Co., Ltd., Taipei, Taiwan). All animal experiments were approved by the Institutional Animal Use and Care Committee, Taipei Tzu Chi Hospital, Taipei, Taiwan (103-IACUC No.020). Care and handling of the rats were performed in accordance with the guidelines of the National Institutes of Health. All rats were supplied with standard laboratory chow and water ad libitum until the day of the experiment.

2.1. Anesthesia and hind limb I/R protocol

All rats were anesthetized with a mixture of zoletil/xylazine (30/10 mg/kg, i.m.) and placed supine on a heating pad. Supplemental doses of the zoletil/xylazine mixture (10/3 mg/kg) were administered hourly until the end of each experiment. To induce hind limb I/R, rubber band tourniquets were applied high around each hind limb for 3 hours followed by removal of the rubber bands for 24 hours, according to previous reports.4, 11 During the reperfusion period, the rats were returned to their cages and supplied with standard laboratory chow and water ad libitum, as mentioned above.

2.2. Experimental protocols

The rats were randomly allocated to the sham operation (Sham), Sham plus cepharanthine (Sham+Cep), limb I/R (I/R), or I/R plus cepharanthine (I/R+Cep) group (n = 6 in each group). The Sham group received sham operation plus vehicle injection [30 μL of dimethyl sulfoxide (DMSO), i.p.; Sigma–Aldrich, St. Louis, MO, USA]. The Sham+Cep group received sham operation plus cepharanthine (10 mg/kg, i.p.; LKT Laboratories, Inc., St. Paul, MN, USA). The I/R group received I/R plus vehicle (30 μL of DMSO, i.p.). The I/R+Cep group received I/R plus cepharanthine (10 mg/kg, i.p.). Administration of vehicle or cepharanthine was performed immediately before reperfusion or at a comparable time point in the Sham groups. The dose of cepharanthine was determined according to a previous report.¹⁰

2.3. Collections of blood and tissue samples and liver function assay

At the end of the reperfusion period, the rats were anesthetized again to facilitate blood drawing. Then, all rats were euthanized with a high-dose pentobarbital (100 mg/kg, i.p.). The liver was removed, snap-frozen, and then stored at −80°C for subsequent analysis.

The blood samples (5 mL) were then centrifuged (1500 g × 5 minutes) to separate plasma. The plasma samples (1 mL) were then analyzed using a chemistry analyzer (Roche Reflotron 1 Chemistry Analyzer; Roche Diagnostic Corp., Indianapolis, IN, USA) to determine the plasma concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

2.4. Assays of nitric oxide and malondialdehyde

The snap-frozen liver tissue samples were processed, as previously reported,¹²to facilitate oxidative status evaluation. In brief, the concentrations of nitrite and nitrate [i.e., the stable nitric oxide (NO) metabolites] of the liver tissue samples were measured using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) to measure the concentrations of reactive nitrogen species. The malondialdehyde (MDA) concentrations of the liver tissue samples were also measured with a thiobarbituric acid reactive substance assay kit (Cayman Chemical) to assay the status of lipid peroxidation.

2.5. Assays of inflammatory molecules

The liver tissue samples were processed, also as previously reported.¹² The inflammatory molecule concentrations of the liver tissue samples, including chemokine (e.g., macrophage inflammatory protein-2, MIP-2), cytokine (e.g., interleukin-6; IL-6) and prostaglandin E₂ (PGE₂), were measured using enzyme-linked immunosorbent assay kits (Enzo Life Science, Farmingdale, NY, USA) according to the manufacturer’s instructions.

2.6. Assay of cyclooxygenase-2 expression

Cyclooxygenase-2 (COX-2) tightly regulates the production of PGE₂.¹³ The expression of COX-2 mRNA in the liver tissue samples was measured using reverse transcription and polymerase chain reaction (RT–PCR). Tissue processing was performed as previously reported.⁴ The primer sequences of COX-2 and β-actin (as an internal standard) and RT–PCR protocols were also adapted from our previous report.⁴ After separation, the PCR-amplified cDNA band densities were quantified using densitometric techniques (Scion Image for Windows, Scion Corp., Frederic, MD, USA).

2.7. Statistical analysis

One-way analysis of variance with Tukey post-hoc test was used for multiple comparisons. Data were presented as means ± standard deviations. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows, SPSS Inc., Chicago, IL, USA) was used for data analysis.

3. Results

3.1. Liver function assay

The plasma levels of AST and ALT of the Sham and Sham+Cep groups were low (Figure 1). As expected, the plasma levels of AST and ALT of the I/R group were significantly higher than those of the Sham group (both p < 0.001; Figure 2). However, the plasma levels of AST and ALT of the I/R+Cep group were significantly lower than those of the I/R group (both p < 0.001; Figure 1).

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Figure 1. Plasma concentrations of AST and ALT. * p < 0.05, versus Sham. ** p < 0.05, I/R+Cep versus I/R. ALT = alanine aminotransferase; AST = aspartate aminotransferase; I/R = the limb ischemia–reperfusion group; I/R+Cep = the I/R-plus-cepharanthine group; Sham = the sham-operation group; Sham+Cep = the Sham-plus-cepharanthine group.

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Figure 2. Hepatic concentrations of NO and MDA. * p < 0.05, versus Sham. ** p < 0.05, I/R+Cep versus I/R. I/R = the limb ischemia–reperfusion group; I/R+Cep = the I/R-plus-cepharanthine group; MDA = malondialdehyde; NO = nitric oxide; Sham = the sham-operation group; Sham+Cep = the Sham-plus-cepharanthine group.

3.2. Oxidative status assays

As expected, the hepatic levels of NO of the Sham and Sham+Cep groups were low, and the hepatic level of NO of the I/R group was significantly higher than that of the Sham group (p < 0.001; Figure 2). By contrast, the hepatic level of NO of the I/R+Cep group was significantly lower than that of the I/R group (p < 0.001; Figure 2).

The hepatic MDA data basically paralleled the hepatic NO data. The hepatic MDA level of the Sham group was low and the hepatic MDA level of the I/R group was significantly higher than that of the Sham group (p < 0.001; Figure 2). Similarly, the hepatic MDA level of the I/R+Cep group was significantly lower than that of the I/R group (p = 0.039; Figure 2). The hepatic MDA levels of the Sham+Cep and I/R+Cep groups were significantly lower than that of the Sham group (p = 0.007 and p = 0.017, respectively; Figure 2).

3.3. Assays of inflammatory molecules

The hepatic levels of MIP-2 and IL-6 of the Sham and Sham+Cep groups were low, and the hepatic levels of MIP-2 and IL-6 of the I/R group were significantly higher than those of the Sham group (both p < 0.001; Figure 3). The hepatic levels of MIP-2 and IL-6 of the I/R+Cep group were significantly lower than those of the I/R group (p < 0.001 and p = 0.039, respectively; Figure 3).

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Figure 3. Hepatic concentrations of MIP-2 and IL-6. * p < 0.05, versus Sham. ** p < 0.05, I/R+Cep versus I/R. IL-6 = interleukin-6; I/R = the limb ischemia–reperfusion group; I/R+Cep = the I/R-plus-cepharanthine group; MIP-2 = macrophage inflammatory protein-2; Sham = the sham-operation group; Sham+Cep = the Sham-plus-cepharanthine group.

Similarly, the hepatic levels of COX-2 mRNA and PGE₂ of the Sham and Sham+Cep groups were low, and the hepatic levels of COX-2 mRNA and PGE₂of the I/R group were significantly higher than those of the Sham group (both p < 0.001; Figure 4). The hepatic levels of COX-2 mRNA and PGE₂ of the I/R+Cep group were also significantly lower than those of the I/R group (p < 0.001 and p = 0.007, respectively; Figure 4).

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Figure 4. Representative gel photography of COX-2/β-actin mRNA and densitometric data of liver and hepatic concentrations of PGE2. * p < 0.05, versus Sham. ** p < 0.05, I/R+Cep versus I/R. COX-2 = cyclooxygenase-2; I/R = the limb ischemia–reperfusion group; I/R+Cep = the I/R-plus-cepharanthine group; PGE2 = prostaglandin E2; Sham = the sham-operation group; Sham+Cep = the Sham-plus-cepharanthine group.

4. Discussion

The data from the present study revealed that limb I/R may create oxidative stress, induce inflammatory response, and eventually cause liver injury in rats. These data are consistent with previous ones.1, 2, 3, 4, 5, 6 The data from the present study also revealed that cepharanthine may reduce oxidative stress, mitigate inflammatory response, and alleviate liver injury induced by limb I/R in rats. As aforementioned, cepharanthine is currently used in clinical settings.8, 9 Therefore, the data from the present study should have profound clinical implications and thus warrant further investigation.

Oxidation and inflammation are crucial in mediating the development of remote organ injury induced by limb I/R.3, 4, 5, 6 Abundant data have shown that limb I/R can cause oxidative stress and elicit inflammatory response in remote organs and subsequently cause remote organ dysfunction and injury.1, 2, 3, 4, 5, 6 This concept is confirmed by the current study. Our data illustrated that rats receiving limb I/R had significant increases in hepatic concentrations of NO, MDA, MIP-2, IL-6, and COX-2/PGE₂. Rats receiving limb I/R also had significant alterations in liver function. Our data illustrated further that rats with lower hepatic concentrations of NO, MDA, MIP-2, IL-6, and COX-2/PGE₂were more likely to have lower levels of functional alterations in the liver. These data thus highlight the crucial roles of oxidation and inflammation in mediating the development of remote organ injury induced by limb I/R.

The data from this study also revealed that cepharanthine may reduce oxidative stress induced by limb I/R. Previous data indicated that cepharanthine possesses potent free-radical scavenging capacity.8, 9, 10Therefore, it is reasonable to observe in our data that cepharanthine could inhibit increases in the levels of free nitrogen species (e.g., NO) and lipid peroxidation in limb I/R rats. In addition, our data revealed that cepharanthine may mitigate inflammatory response induced by limb I/R. One possible mechanism is the expressional regulation of inflammatory molecules. The expression of inflammatory molecules is tightly regulated by the upstream transcription factor nuclear factor-κB (NF-κB). Oxidants are potent activators of NF-κB, and limb I/R can significanly upregulate NF-κB activation.14, 15, 16 As cepharanthine can reduce the production of oxidants, it is reasonable to speculate that cepharanthine may very likely act through oxidant production inhibition and the subsequent mitigation of NF-κB activation to exert its effects on downregulating inflammatory molecule expression. Our recent data that cepharanthine could reduce pulmonary NF-κB expression¹⁷ seems to support this concept.

To control for the vehicle, the rats of the Sham and I/R groups also received intraperitoneal injections of DMSO. However, our data revealed that the hepatic MDA level of the Sham group was significantly higher than that of the Sham+Cep group. These data indicate that DMSO per se may cause oxidative stress and increase lipid peroxidation in rat liver. This concept is supported by previous data that DMSO could increase oxidative stress in yeast.¹⁸Previous data further indicated that the mechanism may involve the effects of DMSO on inhibiting the endogenous antioxidative pathway methionine sulfoxide reductase.¹⁹ The current study revealed that cepharanthine may mitigate the oxidative stress caused by DMSO. The current study also revealed that cepharanthine may inhibit the oxidative stress caused by limb I/R plus DMSO. Together, these data support the potent antioxidative capacity of cepharanthine.

The data from the present study revealed that limb I/R may cause liver injury in rats. However, some previous data indicated otherwise. For instance, a previous report indicated that the liver was not affected by the adverse effects produced by limb I/R in mice.²⁰ As discrepancy is noted, one may argue that the liver injury observed in the present study may be self-limited. The effects of limb I/R are diverse. Brief limb I/R may activate endogenous protective pathways, which may result in the reduction of oxidative stress and the preservation of mitochondrial function.²¹ The protective effects are augmented by repetitive brief limb I/R (e.g., 3 cycles of ischemia for 5–10 minutes followed by reperfusion for 5–10 minutes).22, 23 Based on these findings, ischemic preconditioning (i.e., repetitive brief limb I/R treatment before insult) has thus been developed as an effective therapy against injury induced by ischemia and reperfusion.22, 23 However, the protective effects may wane and gradually shift to adverse ones with longer duration of ischemia. In the aforementioned study, Mansour et al²⁰ reported that limb ischemia for 2 hours followed by reperfusion may induce injury to the lungs, but not to the liver and kidney, in mice. The data from the present study further revealed that limb ischemia for 3 hours may induce liver injury in rats. Using the same model, we have recently shown that limb ischemia for 3 hours followed by reperfusion may also induce injuries to the lungs and kidney.17, 24 Our recent data further revealed that limb ischemia for 3 hours followed by reperfusion may induce injury to lower-limb muscle.²⁴ Collectively, these data highlight the adverse effects of the limb I/R model employed in the present study on inducing injuries to remote organs, including the lungs, liver, and kidney, as well as to local muscle tissues. In line with this notion, we thus believe that the liver injury observed in this study should not be considered as self-limited and proper therapy should be administered.

Certain study limitations do exist and thus need to be addressed. Firstly, the current study investigated only the short-term effects (i.e., 24 hours after reperfusion) of cepharanthine on protecting the liver in limb I/R rats. The long-term effects of cepharanthine in this regard remain to be elucidated. Secondly, whether the protective effects of cepharanthine involve mechanisms other than reducing oxidation and mitigating inflammation also remains unstudied. Thirdly, only one dose of cepharanthine was employed. Whether the protective effects of cepharanthine are dose-dependent remains to be investigated. Similarly, only one timing for cepharanthine administration (i.e., immediately after reperfusion) was employed. Previous data indicated that cepharanthine may improve renal ischemia–reperfusion injury in rats by a pretreatment strategy (i.e., 1 hour before ischemia).²⁵ In line with this notion, we thus speculate that cepharanthine administered before limb ischemia may also exert protective effects against injuries induced by limb I/R. More studies are needed before further conclusions can be reached. Finally, as aforementioned, our recent data indicated that the limb I/R model employed in the present study may cause lower-limb muscle injury.²⁴ We did observe that the rats of the I/R and I/R+Cep groups were not as vigilant as those of the Sham and Sham+Cep groups during reperfusion. As a result, the intake of water and food during reperfusion in the rats of the I/R and I/R+Cep groups was lower than that of the Sham and Sham+Cep groups. To what extent the impacts of the differences in water and food intake may be exerted remains to be elucidated.

In conclusion, cepharanthine alleviates liver injury in a rodent model of limb I/R. The mechanisms may involve reducing oxidation and inflammation.

Acknowledgments

This work was supported by a grant from Taipei Tzu Chi Hospital(TCRD-TPE-1-4-RT-1) awarded to C.J.H.