Study on the Hepatoprotective Effect of Oxalis coriniculata L. and Related Mecha

Ya GAO, Chun CHEN, Kefeng ZHANG, Riming WEI

Guilin Medical University, Guilin 541004, China

Abstract [Objectives] This study aimed to explore the protective effect of Oxalis coriniculata L. on rats with acute liver injury induced by carbon tetrachloride (CCl4) and related mechanism by regulating oxidative stress and the TLR-2 TLR-2/NF-κB signaling pathway. [Methods] A total of 48 female rats were randomly and evenly divided into normal group, model group, silymarin group (0.12 g/kg), and high (16 g/kg), middle (8 g/kg) and low-dose (4 g/kg) O. coriniculata L. groups. The rats in the groups were intragastrically administered with 5 mL/kg of corresponding drugs (equal-volume distilled water for normal group and control group), respectively. The administration was conducted twice a day, for 10 consecutive days. After 2 h of the last administration, the rats in all the groups except the normal group were intraperitoneally injected with 12% carbon tetrachloride (CCl4) olive oil solution (5 mL/kg), respectively to establish liver injury rat models. After 16 h, the eyeball blood of the rats was collected, and their liver tissues were collected for preparation of HE sections. The biochemical indicators detected included aspartate aminotransferase (AST), alanine aminotransferase (ALT), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) content in the serum. The contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the serum were detected by ELISA. The expression of Toll-like receptor-2 (TLR-2) and nuclear factor-κB (NF-κB) in liver tissue was detected using Western blotting. The pathological changes of liver were observed under light microscope. [Results] Compared with the normal group, the ALT, AST activity and MDA, IL-1β, IL-6, TNF-α levels in rat serum significantly increased (P<0.01), the GSH-Px, T-SOD activity in rat serum significantly decreased (P<0.01), and the expression of TLR-2 and NF-κB in liver tissue was up-regulated (P<0.01) in the model group. Compared with the model group, the ALT, AST activity and MDA, IL-1β, IL-6 and TNF-α levels in rat serum reduced (P<0.05, P<0.01), the GSH-Px and T-SOD activity in rat serum increased (P<0.05, P<0.01), and the expression of TLR-2 and NF-κB in liver tissue was down-regulated (P<0.05, P<0.01) in the O. coriniculata L. administration groups. Pathological sections show that O. coriniculata L. had an improving effect on rats with acute liver injury induced by CCl4. [Conclusions] O. coriniculata L. has a good protective effect on rats with acute liver injury induced by CCl4. Its mechanism may be related to inhibition of oxidative stress, inhibition of inflammatory response and regulation of the TLR-2/NF-κB signaling pathway.

Key words Oxalis coriniculata L., Acute liver injury, Oxidative stress, Inflammatory response, TLR-2/NF-κB signaling pathway

1 Introduction

Liver disease is caused by many factors, including hepatitis viruses, chemicals (alcohol and drugs,etc.), metabolic disorders and cholestasis, and has become a serious worldwide disease[1]. The liver is more severely threatened by metabolic toxic damage compared with other tissues and organs in the body. It is the most important organ for metabolism and detoxification[2]. Currently, although many hepatoprotective drugs have been widely used, they all have some potential disadvantages. Seeking safe and effective drugs from Chinese herbal medicines for liver disease has attracted much attention.OxaliscoriniculataL. (Oxalidaceae) is widely distributed in South China, North China, Sichuan, Yunnan and other places. It is cold in nature, sour in taste, metabolized through small intestine and liver, and used as medicine with dried or fresh whole grass, with functions of calming liver, arresting convulsion, removing dampness, diminishing swelling, diminishing inflammation, relieving pain, cooling blood and dissipating blood stasis. It is used to treatment diseases such as jaundice, acute hepatitis and mumps, with certain potential medicinal value[3-4]. Research shows thatO.coriniculataL. contains various ingredients such as flavonoids, vitexin, echinoside, β-tocopherol and carotenoids. Pharmacological studies have shown thatO.coriniculataL. has antibacterial, anti-oxidant, anti-inflammatory and analgesic effects[5-7]. Previous reports on the treatment of liver diseases byO.coriniculataL. were mainly concentrated in folks. It is often used in combination with heat-clearing, dampness-removing, detoxifying, swelling-diminishing and blood circulation-promoting drugs[8]. There has been rare research on the application ofO.coriniculataL. in the treatment of liver injury. The results of a study[9]show thatO.coriniculataL. can reduce the activity of transaminase in mice with liver injury. However, its in-depth pharmacological effects and mechanisms have not been reported. Therefore, in this paper, based on the pathogenesis of liver injury, the protective effect ofO.coriniculataL. on carbon tetrachloride (CCl4)-induced acute liver injury in rats was studied, and the mechanism of action was explored by regulating oxidative stress, inflammatory response and Toll-like receptor-2 (TLR-2)/nuclear transcription factor-κB (NF-κB) signaling pathway.

2 Materials and methods

2.1 AnimalA total of 48 SPF SD rats (female, 200-220 g) were provided by the Experimental Animal Center (SPF grade) of Guilin Medical College, with quality certificate SCXK (Gui) 2007-0001, ethics committee certificate GLMC201703006.

2.2 Drugs and reagentsDried whole plants ofO.coriniculataL., identified by associate professor Zhang Kefeng of the School of Pharmacy, Guilin Medical College, were purchased from the Chinese herbal medicine market in Liuhe Road, Qixing District, Guilin City, Guangxi. A certain amount (2.0 kg) ofO.coriniculataL. was pulverized, soaked in distilled water for 30 min, boiled for 1 h and filtered. The residual was added with an appropriate volume of water and boiled for 30 min. The filtrate of the two times was mixed together, and concentrated under reduced pressure to obtain aqueous extract ofO.coriniculataL., which was stored in a refrigerator at 4 ℃ for use. The drugs and reagents used included CCl4(AR grade, batch No.0710262, Xilong, China), silymarin (batch No.A02140101, Wanbang, China), aspartate aminotransferase (AST) kit (batch No.20170601, Jiancheng, China), alanine aminotransferase (ALT) kit (batch No.20170801, Jiancheng, China), malondialdehyde (MDA) kit (batch No.20170614, Jiancheng, China), total superoxide dismutase (T-SOD) kit (batch No.20170728, Jiancheng, China) and glutathione peroxidase (GSH-Px) kit (batch No.20170721, Jiancheng, China), rat tumor necrosis factor-α (TNF-α) kit (batch No.20170821, Elabscience, China), rat interleukin-1β (IL-1β) kit (batch No.20170821, Elabscience, China), rat interleukin-6 (IL-6) kit (batch No.20170821, Elabscience, China), goat anti-rabbit nuclear factor κB (NF-κB) IgG (batch No.ab59346, Abcam, UK), rabbit anti-mouse TLR-2 IgG (batch No.ab72622, Abcam, UK) and rabbit anti-mouse β-actin (batch No.ab64556, Abcam, UK).

2.3 Apparatus and instrumentsElectronic balance (AUW-220D, Shimadzu, Japan); high-speed refrigerated centrifuge (H2050R, Xiangyi, China); full-wavelength microplate reader (Epoch, BioTek, USA); fluorescence microscope (BX51, Olympus, Japan); transfer box (4530, Bio-Rad, USA); automatic chemiluminescence image analysis system (Tanon5200, Tianneng, China); microtome (KD-3358, Kedi, China); dehydrator (KD-TS3A, Kedi, China); water bath-slide drier (BK-218S, Berna, China); freezer (KD-BL, Kedi, China); paraffin embedding machine (KD-BMII, Kedi, China).

2.4 Methods

2.4.1Grouping, drug administration and modeling. The 48 female rats were randomly and evenly divided into normal group, model group, silymarin group (0.12 g/kg), high-doseO.coriniculataL. group (16 g/kg), middle-doseO.coriniculataL. group (8 g/kg) and low-doseO.coriniculataL. group (4 g/kg). The rats in the silymarin andO.coriniculataL. administration groups were given intragastrically with corresponding drugs according to the dose of 5 mL/kg, and those in the normal group and model group were given with equal volume of distilled water. The administration was carried out twice a day for 10 d. After 2 h of the last administration, the rats in all the groups except the normal group were injected intraperitoneally with 12% CCl4olive oil solution at a dose of 5 mL/kg to induce acute liver injury. Then, the rats were fasted but provided with free access to drinking water. After 16 h, the eyeball blood and liver tissues of the rats were collected for analysis.

2.4.2Preparation of pathological section. The collected liver tissues were fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin, sliced, and stained with hematoxylin-eosin (HE), respectively. Under light microscope, the pathological changes in the liver tissues were observed.

2.4.3Detection of biochemical indicators. After standing at room temperature for 2 h, the blood samples were centrifuged at 4 500 r/min for 15 min at 4 ℃, and the serum was collected and stored in a refrigerator at -20 ℃ for use. According to the instructions of the biochemical kits, the activity of ALT, AST, GSH-Px and T-SOD and the content of MDA in the serum samples were detected. According to the instructions of the ELISA kits, the contents of IL-1β, IL-6 and TNF-α in the serum samples were determined.

2.4.4Western blot analysis of TLR-2 and NF-κB protein expression in liver tissue. A certain amount (about 50 mg) of each of the liver tissues of the same position was added with lysate, ground thoroughly under ice bath and centrifuged. The supernatant was collected. According to the instruction of the BCA kit, the protein content was determined. The supernatant was added with loading buffer, boiled for 5 min and stored in a refrigerator at -20 ℃. Then, 10% separation gel and 5% stacking gel were prepared. The electrophoresis was performed at 80 V first and then at 110 V and ended after the proteins were sufficiently separated. The proteins were transferred to a PVDF membrane under a constant current of 200 mA for 1.5 h and then blocked for 3 h. Then, the membrane was washed with TBST three times with 5 min for each time, added with the primary antibody, incubated at 4 ℃ overnight, washed with TBST three times with 5 min for each time, added with the secondary antibody, incubated at room temperature for 2 h, washed with TBST three times, coated with color developer (solution A∶solution B=1∶1) and placed under a fully automated chemiluminescence imaging system for analysis. Protein gray was analyzed by Quantity One software to calculate the relative expression of the target protein.

3 Results and analysis

3.1 Effects on serum ALT and AST activity in ratsAs shown in Table 1, compared with the normal group, the serum ALT and AST activity of rats in the model group increased (P<0.01); and compared with the model group, the serum ALT and AST activity in the silymarin and high, middle and low-doseO.coriniculataL. groups reduced to different extents (P<0.05,P<0.01).

GroupDose∥g/kgALT∥IU/LAST∥IU/LNormal-47.82±3.9859.74±4.83Model-136.53±18.781)229.66±25.631)Silymarin0.1292.37±10.533)169.37±18.623)O. coriniculata L.1685.31±9.623)157.64±17.383)897.38±11.563)173.59±19.423)4112.29±13.562)188.63±23.573)

Note: Compared with normal group,1)P<0.01; compared with model group,2)P<0.05,3)P<0.01. The same in Table 2-Table 4.

3.2 Effects on serum GSH-Px, T-SOD activity and MDA content in ratsAs shown in Table 2, compared with the normal group, the serum NDA content increased significantly, and the serum GSH-Px and T-SOD activity reduced significantly in the model group (P<0.01); and compared with the model group, the serum MDA content decreased significantly, and the serum GSH-Px and T-SOD activity increased significantly in the silymarin and high, middle and low-doseO.coriniculataL. groups (P<0.05,P<0.01).

3.3 Effects on serum IL-1β, IL-6 and TNF-α contents in ratsCompared with the normal group, the serum IL-1β, IL-6 and TNF-α levels in the model group increased significantly (P<0.01). Compared with the model group, the serum IL-1β, IL-6 and TNF-α levels decreased significantly in the high and middle-doseO.coriniculataL. groups (P<0.05,P<0.01); and the serum IL-1β and IL-6 levels decreased (P<0.05), and the TNF-α level did not change significantly (P>0.05) in the low-doseO.coriniculataL. group (Table 3).

GroupDose∥g/kgT-SOD∥U/LGSH-Px∥U/LMDA∥nmol/mLNormal-143.42±15.53374.89±36.581.36±0.11Model-91.58±10.761)256.72±27.541)2.17±0.191)Silymarin0.12117.59±9.743)294.19±28.543)1.84±0.163)O. coriniculata L.16119.54±12.313)302.46±31.593)1.79±0.163)8112.39±11.783)290.31±31.422)1.86±0.193)4107.38±11.262)281.59±30.211.91±0.172)

GroupDose∥g/kgTNF-α∥pg/mLIL-6∥pg/mLIL-1β∥pg/mLNormal-113.58±13.4662.38±5.7221.57±1.98Model-198.75±23.921)116.54±12.931)39.72±3.531)Silymarin0.12168.73±17.522)93.46±9.693)33.23±2.743)O. coriniculata L.16163.72±15.493)89.67±10.553)30.16±2.653)8172.37±18.642)95.57±10.853)32.98±2.843)4179.73±19.21102.37±11.652)35.43±3.122)

3.4 Effects on the expression of TLR-2 and NF-κB in rat liver tissueThe expression of TLR-2 and NF-κB proteins was low in the liver tissue of normal rats, and was significantly up-regulated in the liver tissue of rat models (P<0.01). Compared with the model group, the expression of TLR-2 and NF-κB in the silymarin and high, middle and low-doseO.coriniculataL. groups was down-regulated to varying extents (P<0.05,P<0.01) (Fig.1, Table 4).

Note: A. normal group; B. model group; C. silymarin group; D. high-doseO.coriniculataL. group (16 g/kg); E. middle-doseO.coriniculataL. group (8 g/kg); F. low-doseO.coriniculataL. group (4 g/kg).

Fig.1 Expression of TLR-2 and NF-κB in liver tissue of rats in various groups

GroupDose∥ g/kgNF-κB/β-actinTLR-2/β-actinNormal-0.412±0.0650.604±0.095Model-1.783±0.2251)1.542±0.1731)Silymarin0.121.265±0.1523)1.176±0.1432)O. coriniculata L.161.148±0.1363)0.957±0.1253)81.326±0.1543)1.147±0.1333)41.393±0.1752)1.253±0.1462)

3.5 Effects on morphology of rat liverThe liver tissue of the rats in the normal group was ruddy, shiny, fine, and grain-free. The liver tissue of rats in the model group was dull, slightly yellowish, slightly expanded, and had a rough surface with different sizes of grains. A small amount of inflammatory exudate was present in the abdominal cavity of certain rats. The pathological changes in the liver tissue of the silymarin and high, middle and low-doseO.coriniculataL. groups were improved to some extent. Among them, the improvement in the high-doseO.coriniculataL. group was the most obvious. Under the light microscope, it could be observed that the liver tissue of rats in the normal group had intact lobules, intact and clear hepatocyte nuclei, no nucleus pyknosis, abundant cytoplasm, and neatly-arranged hepatocyte cords. Compared with the normal group, in the liver tissue of rats in the model group, the hepatocytes were arranged disorderly, a large number of hepatocytes around the central vein and the portal area ruptured and necrosed, and the number of inflammatory cells increased significantly. Compared with the model group, hepatocyte necrosis and inflammatory cell infiltration were significantly improved in the silymarin and high, middle and low-doseO.coriniculataL. groups (Fig.2).

Note: A. normal group; B. model group; C. silymarin group; D. high-doseO.coriniculataL. group (16 g/kg); E. middle-doseO.coriniculataL. group (8 g/kg); F. low-doseO.coriniculataL. group (4 g/kg).

Fig.2 Effects ofOxaliscoriniculataL. on pathological changes in liver tissue of rats(HE, ×200)

4 Discussion

Liver injury caused by CCl4is a chemical liver injury, similar to pathological changes in human liver and reproducible, and it is one of the classic methods of liver damage modeling[10]. CCl4injected into the rat by abdominal cavity can be absorbed into the liver, metabolized by cytochrome P450 (CYP450) to produce trichloromethyl radical (CCl3·) and trichloromethyl peroxy radical (CCl3O2·). This type of free radicals can directly cause lipid peroxidation in the cell membrane, destroy the cell membrane and produce oxidative stress, leading to degeneration and necrosis of hepatocytes[11]. When liver injury occurs, ALT and AST spill into the blood, and their activity increases significantly, so ALT and AST activity can reflect the degree of liver damage[12-13]. The results of this experiment show thatO.coriniculataL. can significantly reduce serum ALT and AST activity in rats with liver injury. At the same time, pathological sections show that the inflammatory cell infiltration and hepatocyte necrosis were relieved in the liver tissue of rats in all theO.coriniculataL. groups, suggesting thatO.coriniculataL. can resist acute liver injury induced by CCl4and has a good protective effect on the liver.

MDA is produced during lipid peroxidation caused by free radicals. It can cause changes in structure of cell membrane and affect its metabolic function, further aggravating liver damage[11]. SOD is an important free radical scavenger in the body. It can clear free radicals in liver tissue, inhibit lipid peroxidation, and protect liver cells. GSH-Px is an important antioxidant enzyme in the body. It can scavenge hydrogen peroxide, inhibit lipid peroxidation, prevent the structure and function of liver cell membrane from being destroyed by free radicals and alleviate liver tissue damage[14-15]. Therefore, detection of MDA content and SOD, GSH-Px activity can reflect the severity of liver damage and level of oxidative stress. The results of this study show that after modeling by CCl4, the serum MDA content increased significantly, and the T-SOD and GSH-Px activity decreased sharply in the model group compared with the normal group, suggesting that CCl4can cause oxidative stress in rats. TheO.coriniculataL. intervention can reduce the content of MDA and enhance the activity of T-SOD and GSH-Px in the serum of rats, suggesting that the inhibition of liver damage byO.coriniculataL. may be achieved by inhibiting the oxidative stress.

TNF-α is an important pro-inflammatory factor. When CCl4induces liver damage, the level of TNF-α in serum and liver tissue will increase significantly. Over-expressed TNF-α stimulates a number of related immune cells, such as monocytes, to produce large amounts of IL-1β, IL-6 and other cytokines, which can in turn promote lymphocyte activation in liver tissue, further inducing chemotaxis of chemokines and promote infiltration of inflammatory cells, leading to local inflammation of the liver tissue[16]. In the inflammatory response system of liver injury, large amounts of TNF-α, IL-1β and IL-6 induce hepatic stellate cells to express TLR-2 receptors[17]. TLR-2 can transduce cellular signals downstream, resulting in overexpression of NF-κB protein, thereby activating the NF-κB signaling pathway[18]. When the NF-κB signaling pathway is overactivated, NF-κB induces the transcription of pro-inflammatory factors such as TNF-α, IL-1β and IL-6, making TNF-α, IL-1β and IL-6 levels further increase, thereby forming a positive feedback loop. Thus, the inflammatory signal is amplified, and the inflammation reaction in liver tissue is aggravated[19-20]. The results of this experiment show thatO.coriniculataL. can down-regulate the levels of TNF-α, IL-1β and IL-6, and inhibit the expression of TLR-2 and NF-κB, suggesting that the inhibition of inflammatory reaction byO.coriniculataL. may be related to the influence on the TLR-2/NF-κB signaling pathway.

In summary,O.coriniculataL. has a significant protective effect on rats with acute liver injury induced by CCl4. The mechanism may be related to inhibition of oxidative stress and inflammatory reaction and influence on the TLR-2/NF-κB signaling pathway, but the specific molecular mechanism remains to be further studied.