Hepatoprotective Activity of Yigan Mingmu Oral Liquid against Isoniazid / Rifampicin-Induced Liver Injuries in Rats

Background: To explore the hepatoprotective effect of Yigan mingmu oral liquid (YGMM) on isoniazid-rifampicin induced liver injury in rats. Methods: Total 38 SD rats were randomly divided into 6 groups including control group, model group, silymarin positive control group, and three YGMM treatment groups. Model group was administered intragastrically with INH (100 mg/kg) and RIF (100 mg/kg) for 14 days. Silymarin group and YGMM treatment groups were administered intragastrically with silymarin (100 mg/kg) and different doses of YGMM (1, 2.5, 5 mg/kg) 2 hours before INH and RIF administration from day 4 to day 14. Results: Rats were sacrificed 16 hours after the last day treatment to determine the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), as well as total bilirubin (TB) content. Oxidative stress was evaluated by measuring total superoxide dismutase (T-SOD) and malondialdehyde (MDA) levels. Histopathological changes in liver tissues were observed under an optical microscope by using hematoxylin and eosin staining. The mice in model groups showed significantly (p < 0.05) increased levels in AST, ALT, ALP, TB and MDA compared to their control groups; and showed significantly (p < 0.05) decreased level in T-SOD. These changes were significantly (p < 0.05) reversed by the YGMM treatments in a dose-dependent manner. Hepatic pathological changes were attenuated or even reversed by silymarin or YGMM treatments. Conclusions: YGMM has a good hepatoprotective activity on isoniazid-rifampicin induced liver injuries in rats. How to cite this paper: Chen, Y.X., Mo, Q.G., Xie, B.B., Ma, B.X., Zang, X.Y., Zhou, G., Cheng, L.Y., Zhou, J.H. and Wang, Y.W. (2018) Hepatoprotective Activity of Yigan Mingmu Oral Liquid against Isoniazid/Rifampicin-Induced Liver Injuries in Rats. Chinese Medicine, 9, 165-178. https://doi.org/10.4236/cm.2018.94010 Received: May 2, 2018 Accepted: December 2, 2018 Published: December 5, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Background
Isoniazid (INH) and rifampicin (RIF) are the most common medications prescribed for tuberculosis treatment, which are the first line drugs used for tuberculosis chemotherapy but also are the most common agents that induce liver disease [1].Combined medication of INH and RIF can greatly increase the incidence of liver injury and disease severity; severe cases may lead to liver necrosis and even liver failure [2] [3].Metabolic site of INH and RIF is the liver, which increases the possibility of liver injury.Liver damage caused by anti-tuberculosis drugs belongs to a drug complication that commonly happened during the anti-tuberculosis treatment [4], as well as an important factor affecting the efficacy of anti-tuberculosis treatment and the prognosis of patients.So far, little is known about the cellular and biochemical mechanisms of INH-or RIF-induced hepatotoxicity, but oxidative stress is considered as one of the mechanisms underlying INH + RIF-induced hepatic injury [5].
As the basis of the classic prescription Siwu Tang [6] and the "Gugen Tang" created by Qing Dynasty Shiduo Chen, Yigan mingmu oral liquid (YGMM) was extended by addition of wolfberry (Lycium barbarum or Lycium chinense) fruit and cassia seed.The medicinal preparation contains 12 traditional Chinese medicines (TCM).Rehmannia glutinosa is taken to enrich the blood, nourish Yin and invigorate the kidney in its traditional use, and has been proved as an effective drug for treatment of diabetes [7] and benefit for cardiovascular system [8].
Angelica sinensis is mainly used for the treatment of women's reproductive problems, such as dysmenorrhea, amenorrhoea, menopause, among others [9].
Modern medical researches demonstrate that it has antioxidant, anti-tumor activity, immunoregulatory activity, neuroprotective effect, etc. [10].White peony root, processed product from the root of Paeonia lactiflora Pall, is traditionally used for alleviating pain, tonifying blood and regulating menstruation.Anti-depressant effect [11], anti-inflammatory and immunomodulatory effects [12] of Paeonia lactiflora Pall have been reported recently.Polygonatum odoratum, Ophiopogon japonicas, and wolfberry fruit have been used for centuries in China as a nourish Qi and Yin, and invigorate the liver and kidney medicine.Polygonatum odoratum has been proved having antioxidative effect [13]; Ophiopogon japonicus has antithrombotic activities [14], anti-inflammatory activities [15], antioxidant and immunoregulatory activity [16].Multiple pharmacological activities of wolfberry fruit have been reported, such as hepatotoxicity [17], hypoglycemic and hypolipidemic effects [18], antioxidant and anti-aging effects [19], and so on.Chrysanthemum flower and cassia seed are traditionally used Y. X.

INH + RIF-Induced Liver Injury
After an acclimatization period of 3 days, 38 rats were randomly divided into 6 groups.Each group contains 6 rats except model group contains 8 rats.
We used body surface area algorithm that was proposed by Shuyun Xu in 1982 [23] to calculate the doses of YGMM.The prescribed oral dose of YGMM is 10 ml once, 3 times a day.We consider that a 60 kg person has a clinical dose of 30 ml/60kg per day, which is converted into a 200 g rat dose.
Rat dose = 0.5 ml/kg × 60 kg × 0.018/0.2kg= 2.7 ml/kg (0.018 is a body surface area factor) Therefore, we chose 2.5 ml/kg as our middle dose, then 5 ml/kg as high dose and 1 ml/kg as low dose for rats.
The design of the animal work refers to Pal et al. [24] and Dong et al. [25] and slightly modified.
Group A (control group) received a daily dose of 0.3% CMC-Na (

Liver Index and Kidney Index Determination
At the end of the experiments, the rats were starved overnight and sacrificed the next day by cervical dislocation.The body weights of rats were recorded before sacrificed and the weights of livers and kidneys were recorded immediately after the rats were sacrificed.Liver index = liver weight/body weight × 100%; Kidney index = kidney weight/body weight × 100%.

Biochemical Determinations in Serum
Blood samples were collected from the heart artery and allowed to coagulate.
The serum was isolated from blood by centrifuging at 3000 rpm for 10 min and conserved at −20˚C for subsequent analysis.The activities of AST, ALT, ALP and the concentration of TB in the serum were determined in accordance with the protocols provided by the manufacturers of the kits.

Measurement of Antioxidant Enzymes and MDA Levels in Liver and Kidney
Liver and kidney samples were dissected and rinsed in ice-cold saline to remove the blood.The liver and kidney homogenates (10%, w/v) were prepared in cold 50 mM potassium phosphate buffer (pH 7.

Histopathological Examination of Liver Tissue
A small piece of the liver tissue was cut from liver and immediately transferred into 10% formalin to fix for 24 h and was embedded in paraffin wax.Then cut the blocks into 5 μm-thick sections in a rotary microtome (RM2125, Leica, US).
The sections were stained with hematoxylin and eosin.Each slice was microscopically (IX51, Olympus, Japan) examined to observe the distribution of histopathological changes.

Statistical Analysis
All data were presented as mean ± S.E.M. Statistical analysis was performed using IBM SPSS 20.0 for Windows.One-way analysis of variance (ANOVA) followed by LSD test was conducted for multiple comparisons.Statistical significance was determined at p < 0.05, p < 0.01, and p < 0.001 levels.the control group.

Liver Index and Kidney Index Determination
The silymarin group and YGMMs had no significant effect on decreasing kidney index compared to model group.With YGMM dose increased, the kidney index gradually decreased, which can be considered that YGMM has an effect on reversing kidney index to control level in a dose-dependent manner.
There is no report on that INH and RIF co-administration can induce the increase of relative kidney weight.This is the first paper to report that INH and RIF co-administration has the effect on inducing kidney hypertrophy statistically.

Biochemical Determinations in Serum
The co-administration of INH and RIF for 14 d induced liver injury, as indicated by a marked increase of serum ALT, AST, ALP, and TB levels (Figures 2(A)-(D)).
The corresponding indicators of the rats treated with silymarin and YGMM were lower than that in the INH + RIF-intoxicated groups.
YGMM pretreatment at 5 ml/kg significantly (p < 0.05, p < 0.001, p < 0.05, p < 0.01, respectively) prevented the elevation of these marker enzymes, and these effects was almost comparable with those observed for corresponding silyma-

Levels of Antioxidant Enzyme and MDA in Liver and Kidney
The INH + RIF-induced liver injury group exhibited significant (p < 0.05) reduction in liver SOD (Figure 3   To prevent the damage caused by oxygen-free radicals, tissues develop an antioxidant defense system, which includes non-enzymatic antioxidants (e.g., glutathione, uric acid, bilirubin, and vitamins C and E) and enzymatic antioxidants, such as SOD, CAT, and glutathione peroxidase.SOD, CAT, and glutathione peroxidase have important physiological functions.The activities of these compounds are easily reduced by lipid peroxide or ROS [30].disulfides [31].The non-enzymatic lipid peroxidation of living organisms is a natural, continually occurring process.However, the levels of non-enzymatic lipid peroxidation can dramatically increase under oxidative stress conditions when ROS and radical production increase [32].Previous studies focused on the toxic effects of lipid-derived carbonyl compounds.For example, compounds such as 4-hydroxy-2(E)-nonenal and MDA have been found to trigger apoptosis, cause mutations, and act as respiratory toxins when exogenously administered to animal cells [33].
The mechanism of INH + RIF-induced hepatotoxicity remains unknown.Sarich et al. [34] have reported that both acetylhydrazine and hydrazine exist during active INH-induced hepatotoxicity in rabbits, thereby indicating that hydrazine is most likely involved in the mechanism of hepatic necrosis.Shih et al. [35] observed

Histopathological Examination of Liver Tissue
For the histopathological observations of the liver sections of INH + RIF-induced liver injury rats, Group A (Figure 5(A)) showed normal lobular architecture and

Conclusion
In this paper, YGMM was evaluated for hepatoprotective activity against INH/RIF-induced liver injuries in rats.YGMM exhibited strong hepatoprotective activity, thereby confirming the traditional use of YGMM as an herbal prescription.The maximum protection against hepatic damage was achieved with the highest tested dose of YGMM (5 ml/kg).The possible YGMM mechanisms of action could include its ability to protect the liver against cellular oxidative damage and to maintain the intracellular level of antioxidant enzymes.Thus, YGMM can be used as a potential hepatoprotective agent or health care product for those people who are under risk of liver injuries while accepting INH and RIF treatment against tuberculosis.
4).The resulting suspension was centrifuged at 5000 rpm for 10 min at 4˚C.The total protein contents were determined by Bradford method.The protein concentration was calculated from the calibration curve: C = 190.38A − 12.40 (C: concentration of protein; A: absorbance at 595 nm; and R 2 = 0.9992).The activities of both SOD and CAT were estimated using commercially available kits.The results were expressed in U per mg protein.The contents of GSH and MDA were expressed in mg per g protein and nmol per mg protein, respectively, based on the protocol provided by the manufacturers of the kits.

Figure 1
Figure1shows the liver index and kidney index of the rats after 14 days' treatment.From the changes in liver index (Figure1(A)), we can see that the liver index of rats in the INH + RIF model group decreased significantly (p < 0.05) compared to the control group.The silymarin group and three YGMM treatment groups (YGMMs) had no significant effect on increasing liver index compared to the model group, but there is a trend showing that liver index can be slightly increased by silymarin and low-dose YGMM treatment compared to the model group.The same observation that INH + RIF co-administration induces the decrease of relative liver weight was reported by Pal et al.[24].From the changes in kidney index (Figure1(B)), we can see that the kidney index of rats in the INH + RIF model group increased significantly (p < 0.05) compared with

Figure 1 .
Figure 1.Effects of YGMM on the (A) liver index and (B) kidney index of INH + RIF-treated rats receiving YGMM or silymarin supplementation.# p < 0.05 indicate significant differences between model group and control group; there are no significant differences between silymarin or YGMMs and model group.
rin-treated groups.As we know, there are many common animal model of acute liver injury, such as modeling with carbon tetrachloride, modeling with alcohol, and modeling with some immune agents.After the introduction of INH in 1969, the problem of INH-induced hepatitis remained.Previous studies postulated that the hepatotoxicity of INH was initiated by cytochrome P450-mediated metabolism of INH to acetylhydrazine and hydrazine[26].RIF is a powerful inducer of mixed function oxidase in man and rats and contributes to the hepatotoxicity of INH by enhancing the production of toxic metabolites[27].The combination of INH and RIF as antitubercular drugs has a long history stated that INH and RIF administered in combination resulted in more severe effects than when INH was administered alone[28].Moreover, elevated transaminase levels were observed in clinical cases administered with INH and RIF[29].Y. X. Chen et al.DOI: 10.4236/cm.2018.94010171 Chinese Medicine
(A)) and CAT (Figure 3(B)) activities, produced a significant consumption of liver GSH (Figure 3(C)) content, and showed a remarkable promotion of liver MDA (Figure 3(D)) content compared with the control group.The hepatoprotective effect of YGMM at the highest dose significantly (p < 0.05) elevated the reduction in SOD, CAT, and GSH concentrations and reduced the increase in MDA level compared to the model group.The highest dose YGMM showed the competitive effect on reversing the SOD, CAT, and GSH levels and better effect on decreasing MDA level compared to silymarin's treatment.The INH + RIF administrated group exhibited significant (p < 0.05) reduction in kidney SOD (Figure 4(A)) and CAT (Figure 4(B)) activities, produced a significant consumption of kidney GSH (Figure 4(C)) content, and showed a remarkable promotion of kidney MDA (Figure 4(D)).An unexpected protective effect was observed in kidney tissues.The highest dose of YGMM significantly (p < 0.05) maintained the normal levels of CAT, GSH, and MDA (Figures 4(B)-(D)),

Figure 4 .
Figure 4.The (A) T-SOD, (B) CAT, (C) GSH activities, and (D) the levels of MDA in the kidney of INH + RIF-treated rats receiving YGMM or silymarin supplementation.# p < 0.05 indicate significant differences between model group and control group; *p < 0.05 and **p < 0.01 indicate significant differences between silymarin or YGMMs and model group.

2. Materials and Methods 2.1. Chemicals
[22] et al.The hepatoprotective effect of Chrysanthemum flower has been proved by Jeong et al.[20].Chrysanthemum flower and cassia seed are demonstrated to have antimicrobial activity respectively by scientific research[21][22].All the herbs mentioned above are the main constitution of the YGMM, along with Ligusticum chuanxiong, Anemone altaica, Citrus reticulate, and Bupleurum spp., which are the complementary constitution DOI: 10.4236/cm.2018.94010167 Chinese Medicine for clearing liver and improving eyesight.
SOD converts O 2 into H 2 O 2 , and CAT metabolizes H 2 O 2 to H 2 O and O 2 .GSH acts as an antioxidant in conjunction with various enzymatic processes that reduce hydrogen peroxide and hydroperoxide by oxidizing GSH to glutathione disulfide and other mixed