Baicalein Preconditioning Modulates Hepatocellular Injury following Liver Ischemia and Reperfusion in Rats via Anti-Inflammatory and Antioxidant Signaling

Liver transplantation is the only established treatment for acute liver failure caused by acute drug toxicity, viral hepatitis, or end-stage liver disease. Hepatic ischemia/reperfusion (I/R) is a frequent and major complication during liver transplantation. It is a series of complicated cellular events that occur upon restoration of hepatic blood flow (reperfusion) after a period of ischemia [1]. There are multiple mechanisms that are involved in the pathophysiology of I/R injury, one of them involving the deleterious effect of reactive oxygen species (ROS) and other free radicals. In addition, inflammatory reactions are mediated through transcription of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL-1, IL-6) [2-4]. Previous studies have demonstrated that oxidative stress and inflammation play an important role in the pathogenesis of I/R [2]. Therefore, the use of antioxidants or anti-inflammatory agents may hinder or even prevent the deleterious effects of I/R injury.

Baicalein (5,6,7-trihydroxyflavone) is one of the major flavonoids originally extracted from the roots of the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi). Baicalein is a potent antioxidant and free radical scavenger (hydroxyl radicals) and has been regarded as a specific 12/15-lipoxygenase inhibitor and a good xanthine oxidase inhibitor [5-9]. Baicalein also has anti-inflammatory properties since it has been shown to antagonize the expression of adhesion molecules induced by IL-1β and TNF-α [10].

Previous studies showed baicalein to exert a protective effect on neurons against neuronal injury secondary to ischemia insult [11, 12] through the inhibition of inflammatory mediators and ROS [13]. Furthermore, a recent study showed that baicalein injection 30 min before reperfusion promoted recovery from renal injury and alleviated kidney injury in a rat model of renal I/R via antioxidant and anti-inflammatory effects [14]. Previous studies also demonstrated that baicalein has hepatoprotective effects on acute liver failure induced by D-galactosamine/lipopolysaccharide [15]. Moreover, a recent study by Liu et al. [16] showed that baicalein pretreatment protects against hepatic I/R injury in mice via an anti-inflammatory effect. Thus, the present study aimed to investigate the effects of baicalein preconditioning on liver I/R in rats and the underlying mechanisms.

Baicalein was purchased from Shannxi, China. Its purity was 98% tested by high-performance liquid chromatography. Dimethyl sulfoxide (DMSO) and other chemicals were purchased from Sigma-Aldrich.

Twenty-four adult male Wistar rats (200–220 g; Zagazig University, Zagazig, Egypt) were used in the current study. All rats were kept on a 12-h light/dark regime, with free access to food and water.

Rats were randomly assigned to three groups (8 rats in each group), and drug or solvent was administrated intraperitoneally 30 min before ischemia. All rats were anesthetized by intraperitoneal injection of ketamine (75 mg/kg). Group 1 rats (sham group) received an equal volume of DMSO vehicle and were then anesthetized; the portal vein and bile duct were exposed but not occluded. Group 2 rats (I/R group) received an equal volume of DMSO and were then subjected to partial liver ischemia (70%) followed by reperfusion. Ischemia was induced by occluding the hepatic portal vein and the bile duct with a traumatic vascular clamp for 45 min, then the clamp was removed to start reperfusion for 1 h. Group 3 rats (baicalein group) received 300 mg/kg baicalein dissolved in DMSO and were then intraperitoneally injected a single dose 30 min before ischemia. Although each experimental group consisted of 8 rats, statistical analysis was performed on 6 rats as 2 rats died during the surgical procedure.

Blood was collected from the retro-orbital plexus and centrifuged (3,000 g, 4°C, 20 min) for separation of serum. The serum was used to analyze liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and gamma-glutamyl transferase [GGT]) and lactate dehydrogenase (LDH) enzyme. Then animals were sacrificed, and the liver was separated and washed off with cold saline. Livers were divided into two parts: one part was immediately flash-frozen in liquid nitrogen and kept at –80°C for measurement of tissue parameters, and the other part was kept in 10% formalin for histopathological examination.

The serum ALT, AST, and GGT enzyme activities as well as LDH level were measured using commercially available analytical kits (Biodiagnostic Co., Egypt).

The TNF-α level in liver homogenate was detected by ELISA according to the method of Aggarwal and Vilcek [17] using an ELISA kit (Quantikine, USA). Briefly, 50 µL of assay diluent was added to each well. Then, 50 µL of standard, control, or sample was added. The plate was covered with a plate sealer and incubated at room temperature for 2 h. Then we aspirated each well and washed it, repeating the process four times for a total of five washes. We added 100 µL of conjugate to each well. Then we covered it with a new plate sealer and incubated it at room temperature for 2 h. After that it was aspirated and washed five times. We added 100 µL of substrate solution to each well and incubated it at room temperature for 30 min. Finally, 100 µL of stop solution was added to each well. The absorbance was read at 450 nm within 30 min. The IL-10 level in liver homogenate was detected similarly by ELISA according to the method of Moore et al. [18] using an ELISA kit (Bio Vendor, Germany).

Total RNA was extracted from liver tissue homogenate using the SV Total RNA Isolation system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The extracted RNA was reverse transcribed into cDNA using an RT-PCR kit (Stratagene, USA) according to the manufacturer’s instruction. The real-time PCR reaction mixture was composed of 25 mL SYBR Green Mix (2×), 0.5 mL cDNA, 2 mL of each primer pair mix (5 pmol/mL each primer), and H₂O to 50 mL. The PCR program used to amplify cDNA consisted of 120 s at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 94°C, 30 s at 60°C and 30 s at 72°C, followed by 10 min at 72°C. The real-time PCR result was analyzed with the step PEApplied Biosystems (Perkin Elmer) software and the data were expressed in cycle threshold. Target gene expression was assessed and related to reference gene (β-actin) [19].

The generation of ROS in response to hepatic I/R injury was evaluated in liver tissues by the measurement of manganese superoxide dismutase (MnSOD) content, glutathione peroxidase (GPx), and the lipid peroxidation product malondialdehyde (MDA). MDA, MnSOD, and GPx were measured in tissue homogenate photometrically (spectrophotometer, Jenway®, UK) according to Nishikimi et al. [20] and Ohkawa et al. [21], respectively.

Caspase-3 activity was quantified by proteolytic cleavage of the fluorogenic substrate 7-amino-4-trifluro-methylcoumarin-conjugated Asp-Glu-Val-Asp tetrapeptide (AMC-DEVD) [22]. Liver tissue (20 mg) was homogenized in lysis buffer at 4°C. After -freezing and thawing four times, the lysates were centrifuged at 15,000 rpm for 10 min and the supernatant was collected. Then caspase-3 activity was measured with a fluorescence spectrophotometer (Hitachi F-3010, Japan) using a caspase-3 fluorescence kit (Sigma Co., USA) according to the manufacturer’s instructions. The caspase-3 activity unit was nmol AMC release/h/mg liver tissue [23].

Liver tissues were fixed in 10% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and then examined under a light microscope for determination of histopathological changes. The histological analysis was performed by a person blinded to treatment (Fig. 1).

Values were expressed as mean ± standard deviation. To analyze the differences between groups, statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test using a computer software (GraphPad, Prism 5, USA). A p value <0.05 was considered significant.

The serum ALT, AST, and GGT enzyme activities in the different groups are shown in Figure 2. At 60 min after reperfusion, serum ALT, AST, and GGT enzyme activities were significantly higher in the I/R group than in the sham group. Pretreatment with baicalein showed a significant decrease in the activities of serum ALT, AST, and GGT compared to the I/R group (p < 0.05).

Histopathological examination of hepatic tissues confirmed the serum transaminases estimation of liver damage. Severe liver injury indicated by hepatocellular degeneration, pyknosis, sinusoidal congestion, and neutrophil infiltration was present in the I/R group compared to the sham group, which was normal. Baicalein preconditioning showed less damage and more recovery from I/R injury (Table 1).

Effect of Baicalein on NF-κB Expression. The expression of NF-κB in the livers of experimental rats was examined by real-time RT-PCR. The mRNA expression of NF-κB was significantly increased in the livers of rats in the I/R group when compared to the sham group. A significant reduction in NF-κB mRNA expression level in the baicalein preconditioning group was observed when compared to the I/R group (p < 0.05; Fig. 3).

Effect of Baicalein on TNF-α. The level of the inflammatory marker TNF-α in the livers of experimental rats was examined by ELISA. Compared to the sham group, the TNF-α level was significantly increased in the livers of rats in the I/R group. Compared to the I/R group, a significant reduction in TNF-α level was observed in the baicalein preconditioning group (p < 0.05; Table 2).

Effect of Baicalein on IL-10. The level of the anti-inflammatory marker IL-10 in the livers of experimental rats was examined by ELISA. Compared to the sham group, the IL-10 level was significantly decreased in the livers of rats in the I/R group. Compared to the I/R group, a significant elevation of IL-10 level was observed in the baicalein preconditioning group (p < 0.05; Table 2).

Effect of Baicalein on the TNF-α/IL-10 Ratio. The ratio of the inflammatory marker TNF-α to the anti-inflammatory marker IL-10 in the livers of experimental rats was calculated. Compared to the sham group, the TNF-α/IL-10 ratio was significantly increased in the livers of rats in the I/R group (p < 0.05). Compared to the I/R group, a significant reduction in this ratio was observed in the baicalein preconditioning group (p < 0.05; Table 2).

Since oxidative stress plays an important role in hepatic I/R injury and baicalein is a potent antioxidant, we examined the changes in lipid peroxidation and the level of endogenous antioxidant enzymes in the liver upon baicalein pretreatment.

Effect of Baicalein on Lipid Peroxidation. As shown in Figure 4a, in the present study, compared to the sham group, the lipid peroxidation product MDA was significantly increased in the livers of rats in the I/R group. Baicalein preconditioning significantly blunted the increased lipid peroxidation in the liver compared to the I/R group (p < 0.05).

Effect of Baicalein on Antioxidant Enzymes (MnSOD, GPx). The results in Figure 4b and c show that the MnSOD and GPX activities were significantly reduced in the I/R group compared to the sham group. Baicalein preconditioning significantly restored the activities of endogenous antioxidant enzymes in the liver compared to the I/R group (p < 0.05).

We next asked whether baicalein preconditioning affects apoptotic or necrotic cell death in the liver. I/R injury has been shown to increase caspase-3 activity, which in turn induces apoptotic cell death in the liver. As shown in Figure 5, caspase-3 and LDH cytolytic enzyme activities were significantly increased in the I/R group compared with the sham group. Baicalein preconditioning significantly suppressed caspase-3 activity and thus reduced apoptotic cell death in the liver compared to the I/R group. Furthermore, baicalein significantly reduced LDH activity and necrotic cell death in liver tissues compared to the I/R group (p < 0.05).

In this study, we found that baicalein preconditioning protects the liver against I/R injury by inhibition of ROS production, preservation of endogenous antioxidants, and suppression of inflammatory response. Moreover, our results showed that in livers subjected to I/R, baicalein can modulate apoptosis/necrosis and activate cell survival pathways against oxidative injury and inflammation. Those protective effects are related to increase in endogenous antioxidants, increase in anti-inflammatory cytokine, IL-10, reduction in leukocyte infiltration, and the associated increase in NF-κB mRNA transcription and production of TNF-α and reduction in the TNF-α/IL-10 ratio.

Oxidative stress is the milestone of several liver diseases and modulates acute and chronic cell injury in the liver [24, 25]. ROS including hydrogen peroxide, superoxide anion, and other free radicals are generated during reperfusion of ischemic liver [26]. The damage created by oxidative stress may be direct or indirect. Directly, ROS can damage hepatocytes by many mechanisms, including lipid peroxidation, DNA oxidation, and enzyme denaturation [27-29]. They also may act indirectly by acting as signaling molecules that upregulate NF-κB expression, activating neutrophil accumulation in the liver and releasing TNF-α. This may lead to not only inflammation and necrosis, but also apoptosis [30]. In the present study, 30-min pretreatment with baicalein prevented loss of cell viability and reduced lipid peroxidation while preventing reduction in the activity of both MnSOD and GPx enzymes. MnSOD is considered an important antioxidant enzyme in the mitochondria which protects against oxidative stress. Previous studies showed that baicalein restored hydrogen peroxide-induced reduction in MnSOD protein expression and activity in mitochondria by activating the transcription factor NF-E2-related factor 2 (Nrf2), which is a critical regulator of MnSOD in mitochondria [31]. On the other hand, GPx reduces lipid hydroperoxides to their corresponding alcohols and reduces free hydrogen peroxide to water. Previous studies showed that baicalein abolished cognitive deficits in rats by increasing the activity of GPx, superoxide dismutase, and other antioxidants, enzymes, and anti-inflammatory effects [32]. Moreover, baicalein showed protection against I/R injury in cardiomyocytes via its scavenging capacity of free radicals [33]. Although we did not measure the direct free radical scavenging activity of baicalein in the present study, being a flavonoid, baicalein possess free radical scavenging and lipid peroxidase-inhibiting activity [6, 34]. As mentioned previously, ROS during reperfusion act as signaling molecules that upregulate mRNA expression of NF-κB. NF-κB is a transcription factor located at the cytoplasm in inactive form. During I/R, it is activated by phosphorylation and translocated to the nucleus where it induces gene expression of several genes including TNF-α [30]. In our study we demonstrated that following I/R injury, the mRNA expression of NF-κB is increased, which is in accordance with previous studies. We also showed that the TNF-α level in the liver tissues was elevated, which is a direct consequence of increased NF-κB. By binding with TNF-α type 1 receptors, TNF-α causes further phosphorylation and activation of NF-κB, creating a vicious cycle, and increases the extent of damage. We showed previously that during hepatic I/R injury not only the expression of TNF-α is increased, but also upregulation of TNF-α type 1 receptors takes place [35]. Activation of NF-κB increases the expression of ICAM-1 and other adhesion molecules that increase the recruitment of inflammatory cells to the liver following reperfusion [36]. The infiltrating cells also produces more TNF-α and other proinflammatory cytokines which aggravate inflammation and hepatic injury. We showed in our study that there was an increase in inflammatory cell infiltration following I/R that confirmed the previous reports. Although TNF-α plays an important role in I/R injury in the liver, we showed in our previous study that it is not the only player in mediating hepatic damage and that neutralizing TNF-α by infliximab, a monoclonal antibody, partially protects against the deleterious effects of I/R [35].

IL-10 is an anti-inflammatory cytokine. It produces its anti-inflammatory effects by binding to IL-10 receptors, which induces STAT3 signaling with subsequent increases in cell survival [37]. Previous studies showed that IL-10 suppresses NF-κB signaling and promotes hepatic cell survival [38, 39]. Reduction in anti-inflammatory cytokines such as IL-10 seems to play a crucial role in I/R-induced hepatic damage. We demonstrated in the present study that I/R was associated with a reduction in the level of IL-10 in hepatic tissues. A recent study showed that exogenous administration of IL-10 increased the survival of ob/ob animals at 24 h after I/R and significantly decreased serum ALT levels [40]. In our study it seems that the downregulation of NF-κB by baicalein was mainly mediated via the elevation of IL-10 level. This may be responsible for the reduction in TNF-α level and neutrophil infiltration observed in baicalein-treated rats. I/R also increases the TNF-α/IL-10 ratio, indicating an imbalance between inflammatory and anti-inflammatory cytokines favoring hepatic inflammation and cell damage. Baicalein preconditioning reverses these deleterious changes and decreased the TNF-α/IL-10 ratio. A recent study showed that baicalein pretreatment protects mice against I/R injury by inhibition of the NF-κB pathway, which confirmed our present findings [16]. Previous studies did not investigate the effect of baicalein, anti-inflammatory cytokines, or oxidant signaling in I/R models. In our study we demonstrated that baicalein preconditioning using a 300 mg/kg dose not only suppressed the hepatic inflammation following I/R, but also alleviated oxidative stress, restored endogenous antioxidants, and increased the anti-inflammatory cytokine IL-10.

Apoptosis and necrosis are central mechanisms of cell death in liver IR injury that directly indicate cell death conditions. This study analyzed apoptotic cells in ischemic liver 1 h after reperfusion. Histopathological examination of liver tissues showed hepatocyte degeneration and pyknosis following I/R. Then, the caspase-3 activity of liver tissue was also examined 1 h after reperfusion, which also reflected the condition of apoptotic cells in ischemic liver. In line with the histopathological examination, I/R significantly increased caspase-3 activity after reperfusion. We also showed that the cytolytic enzyme activity LDH was increased following reperfusion, indicating cytolysis. Baicalein preconditioning inhibits caspase-3 as well as LDH activity and reduces pyknosis and hepatocyte degeneration. The antiapoptotic effect of baicalein may be attributed to its antioxidant and anti-inflammatory effects.

Taken together, the present study provides evidence that baicalein preconditioning exerts hepatoprotective effects through inhibiting inflammatory and oxidant signaling and promoting IL-10 in a rat model of hepatic I/R injury. Baicalein could be a promising drug for patient having undergone liver transplantation in clinical settings.

The authors would like to thank Prof. L. Rashed (Biochemistry Department, Faculty of Medicine, Cairo University, Egypt) for assistance in performing the PCR technique and Dr. Dr. A.A. Abuel-Atta, Faculty of Veterinary Medicine, Zagazig University for histopathological examination of livers.

The protocol was approved by the institutional animal care and use committee and the local experimental ethics committee. The authors have no ethical conflicts to disclose.

The authors have no conflicts of interest to declare.

This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

S. Gamal contributed to the experimental design, performed the experiments, conceived the study, oversaw all data collection and analysis, and drafted the manuscript. H.M. El-Fayoumi contributed to the experimental design and manuscript preparation. M.F. Mahmoud contributed to the experimental design, data analysis and interpretation, and manuscript preparation. All authors read and approved the final manuscript.