Dendrobine attenuates isoniazid- and rifampicin-induced liver injury by inhibiting miR-295-5p

Abstract

The present study aims to investigate the protective effects of Dendrobine and its underlying mechanisms on liver injury induced by isoniazid (INH) and rifampicin (RIF). A mouse model of liver injury was induced by intragastrically administration of 100 mg/kg INH and 100 mg/kg RIF for 14 days. The mice were intragastrically administrated with Dendrobine (50, 100, and 200 mg/kg) before the administration of INH and RIF. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Oxidative stress markers including glutathione, superoxide dismutase, and malondialdehyde in the liver were measured and liver histopathological examinations were performed. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were applied to determine the mRNA and protein expressions, respectively. Luciferase reporter assay was used to evaluate the interactions between miR-295-5p and CYP1A2. Dendrobine significantly decreased serum ALT and AST and inhibited the liver index and ameliorated the liver histological changes induced by INH and RIF. Besides, Dendrobine also regulated oxidative stress status in the liver by the regulation of CYP1A2. Moreover, mmu-miR-295-5p was identified to target CYP1A2 and to regulate the expression of CYP1A2. In summary, Dendrobine ameliorated INH and RIF induced mouse liver injury by miR-295-5p-mediated CYP1A2 expression.

Keywords

Liver injury Dendrobine oxidative stress CYP1A2 miR-295-5p

Introduction

Drug-induced liver injury is one of the most frequent causes of liver failure. There are approximately 10% of acute liver failure caused by prescription.^1,2 For instance, isoniazid (INH) is an antibiotic used to treat and/or prevent tuberculosis. However, it causes mild liver injury and its underlying mechanism associated with the production of antibodies against cytochromes P450 (CYP450), an important enzyme family for oxidization and clearance for various compounds.^3,4 Additionally, rifampicin (RIF) is another antibiotic that is used for the treatment of tuberculosis. It is metabolized into desacetyl RIF by CYP3A4 and multidrug resistance-associated protein 2. One of the side effects caused by the idiosyncratic metabolic products of RIF is liver injury.⁵ Moreover, when RIF is combined with other antituberculosis drugs such as INH in the treatment of tuberculosis, they are more likely to cause severe liver injury for patients.³ It is known that both INH and RIF are first-line medications used in the treatment of tuberculosis. However, their uses cause liver toxicities including hepatitis and liver failure.⁶ Thus, it is worthwhile to discover drug candidates to reverse liver toxicities induced by INH and/or RIF.

Dendrobium nobile has been extensively used as a traditional Chinese medicine for many years which manifest a diversity of effects including anti-inflammatory and pain relief.^7,8 D. nobile has drawn much attention in recent years. There are more than 100 compounds which were isolated and identified from the medicinal parts of Dendrobium species.^9
–11 Dendrobine is an alkaloid and firstly was isolated from D. nobile by Yusuki and colleagues in 1932.¹² Chen and colleagues in 1935 identified 15 mg/kg Dendrobine with weak analgesic effect on mice and 8.5 mg/kg Dendrobine with antipyretic effects on rabbits.¹³ In 1983, Kudo and colleagues identified Dendrobine as an antagonist of β-alanine.¹⁴ More recent studies have shed light on anti-virus activities and hypertension effects of Dendrobine.^15,16 In 2017, Li and colleagues have demonstrated that Dendrobine inhibits influenza A virus by the regulation of virus replication.¹⁵ In 2018, Liu and colleagues have reported the inhibitory effects of Dendrobine on the rat cardiac fibroblast by regulating the nuclear factor-κB signaling pathways.¹⁶

To our best knowledge, it is still unknown whether Dendrobine has protective effects on liver injury. Thus, in the present study, for the first time, we aim to investigate the protective effects of Dendrobine on liver injury induced by INH and RIF. Notably, our results showed liver protective effects of Dendrobine on a mouse model of liver injury. Additionally, the results showed that the treatment of Dendrobine affected oxidative stress in the liver by the regulation of CYP1A2.

MicroRNAs (miRNAs), 20- to 22-nucleotide noncoding RNAs, have been identified to be involved in many cellular events by the regulation of genes.¹⁷ A recent study has identified that miR-295-5p is involved in the nonalcoholic fatty liver disease and is associated with triglyceride accumulation.¹⁸ Interestingly, in the present study, for the first time, we identified miR-295-5p is also a target of Dendrobine for the regulation of CYP1A2. Dendrobine ameliorated INH and RIF induced mouse liver injury by the regulation of miR-295-5p-mediated CYP1A2 expression.

Materials and methods

Animals and experimental design

ICR (C57BL6) mice were purchased from Shanghai Laboratory Animal Co., Ltd (SLAC, Shanghai, China). The mice were kept under temperature- and humidity-controlled condition (the temperature at 22–24°C and humidity at 60 ± 5%). All animal protocols were conducted and approved by the ethical committee of Shijiazhuang No. 5 City Hospital.

After adaptive feeding for 1 week, the mice were divided into five groups. In the control group, the mice were administrated with a saline solution for 14 days. In the INH plus RIF group, the mice were administrated with 100 mg/kg INH and 100 mg/kg RIF for 14 days. In the Dendrobine (Den) groups, the mice were administrated with Dendrobine at doses of 200, 100, or 50 mg/kg 2 h before the administration of INH (100 mg/kg) plus RIF (100 mg/kg). The administration of Dendrobine was continuous for 14 days. At the end of the experimental period, mice were sacrificed after the last administration for 16 h. To calculate the liver indexes of each group. The body weight was recorded and the liver was collected and weighted. The liver index was calculated using the following formula: liver index = liver weight/body weight. Additionally, blood was collected and serum was separated for the following assays.

Measurement of alanine aminotransferase and aspartate aminotransferase

After the mice were sacrificed, blood was collected and serum was separated. Next, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using commercial kits according to the document of the manufacturers.

Histopathological analysis

At the end of the experimental period, the mice were sacrificed and the liver was collected. The liver was fixed in 10% formalin solution following by the embedding in paraffin. After that, hematoxylin and eosin (H&E) staining was performed and the slides were observed under a microscope.

Evaluation of oxidative stress-related markers

At the end of the experimental period, the mice were sacrificed and the liver was collected. The levels of oxidative stress-related markers in the liver including glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured using commercial kits according to the documents of the manufacturers.

Cell line and cell culture

AML12 cell line was purchased from the Cell Bank of the Chinese Academic of Sciences (Shanghai, China). The cells were cultured in DMEM:F12 medium supplemented with 10% fetal bovine serum (Life Technologies, Gaithersburg, Maryland, USA), 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone at 37°C in the presence of 5% CO₂ at constant humidity.

Western blot

The protein was extracted according to the previous methods.^19,20 In brief, a cold radioimmunoprecipitation buffer containing protease inhibitor was used to lyse the liver sample. After that, the extraction buffer was centrifuged at 13,000 × g for 10 min to remove the sample debris and other insoluble materials. The BCA protein assay kits were applied to qualify the concentrations of extracted proteins.

An equal amount of proteins was loaded and separated using the 10% sodium dodecyl sulfate gel. After that, the gel was transferred to a polyvinylidene fluoride membrane, which was blocked with 5% nonfat milk at room temperature for 2 h. Next, a primary antibody was used to incubate with the membrane at 4°C overnight. Appropriated secondary antibodies conjugated with horse radish peroxidase were used. glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The imaging system was applied to qualify the expressions of CYP1A2. CYP1A2 antibody was purchased from Sigma (St. Louis, Missouri, USA).

Prediction of target mmu-microRNAs

To predict the possible target mmu-miRNAs of CYP1A2, the online tool Targetscan (http://www.targetscan.org) was applied. Besides, the levels of predicted miRNAs in the liver were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The procedure of qRT-PCR is shown as follows.

Quantitative reverse transcription polymerase chain reaction

RNA extraction kit was used to isolate RNA from the liver, according to the manufacturer’s instructions. Reverse transcriptase was used in the RT reaction. The sequences of primers are as follows. mmu-miR-7116-3p forward: 5′-aga aca gtg aag aca tca gga-3′ and reverse: 5′-cca gtt ttt ttt ttt ttt tct gag aag′; mmu-miR-6951-3p forward: 5′-ttt gtg tga tta aag tat tag aag tat tga aat tct gag ttt tgc t-3′ and reverse: 5′-ggt cca gtt ttt ttt ttt ttt tct gt-3′; mmu-miR-6928-3p forward: 5′-att tgc atg gac tgc act aag aca ctt cag tgt tcc taa agc tgt tcc tgt-3′ and reverse: 5′-cag ttt ttt ttt ttt ttt ctg gag ag-3′; mmu-miR-295-5p forward: 5′-aaa tgt ggg gca cac ttc tgg act gta cat aga aag tgc tac tac ttt tga gt-3′ and reverse: 5′-cca gtt ttt ttt ttt ttt tgg aga g-3′; mmu-miR-292a-5p forward: 5′-tac tca aac tgg ggg ctc ttt tgg att ttc atc gga aga aaa gtg ccg cca ggt-3′ and reverse: 5′-agg tcc agt ttt ttt ttt ttt ttc aa-3′; mmu-miR-290a-5p forward: 5′-ggt act caa act atg ggg gca ctt ttt ttt ttc t-3′ and reverse: 5′-gtc cag ttt ttt ttt ttt ttt ctc aac-3′; mmu-miR-186-5p forward: 5′-gaa ttc tcc ttt tgg gct ttc tca ttt tat ttt aag ccc taa ggt ga-3′ and reverse: 5′-gtc cag ttt ttt ttt ttt ttt act tcc-3′; mmu-miR-7118-5p forward: 5′-aag gcg gga gag gga aca gaa caa cag cta act cta cgt cct cct-3′ and reverse: 5′-gtc cag ttt ttt ttt ttt ttt ctg tg-3′; mmu-miR-183-3p forward: 5′-cac tgg tag aat tca ctg tga aca gtc tca gtc-3′ and reverse: 5′-ggt cca gtt ttt ttt ttt ttt tct g-3′; mmu-miR-143-3p forward: 5′-agt gct gca tct ctg gtc agt tgg gag tct gag atg aag cac tgt agc tc-3′ and reverse: 5′-ggt cca gtt ttt ttt ttt ttt tcc t-3′; mmu-miR-463-3p forward: 5′-ttt gtt gtc cat cat gta aaa cat aaa tga tga tag aca cca tat aag gta gag gaa ggt-3′ and reverse: 5′-ggt cca gtt ttt ttt ttt ttt tag tga a-3′. The melt curves were used to analyze the accuracy. The expressions of each gene were calculated using 2^−ΔΔCt values. The mRNA expression values of target genes were normalized to that of GAPDH.

Luciferase reporter assay

Luciferase reporter assay was applied to investigate the interactions between CYP1A2 and mmu-miR-295-5p. In brief, when the cells reach 60–70% confluency, the cells were co-transfected with plasmids containing a sequence of CYP1A2 3′UTR wide type (WT) or CYP1A2 3′UTR mutation (MYT) and mmu-miR-295-5p or scramble sequence. The activities of luciferase were determined after the transfection of 24 h.

Statistical analysis

Data were shown as mean ± S.D. One-way analysis of variance with multiple comparisons and Student–Newman–Keuls test were performed. A p value that less than 0.05 was thought as a statistical significance between the two groups.

Results

Effects of Dendrobine on the serum ALT and AST in the liver injury mouse model induced by INH plus RIF

We first measured the levels of serum ALT and AST. The results demonstrated that the levels of ALT and AST were significantly increased in the INH plus RIF group as compared to the control group (Figure 1(a) and (b)). However, the levels of ALT and AST in the Dendrobine-treated groups are decreased as compared to the INH plus RIF group. In addition, no statistical difference was observed between Dendrobine (at 50 mg/kg) group and INH plus RIF group. Interestingly, administration of Dendrobine at doses of 100 mg/kg and 200 mg/kg significantly decreased the levels of ALT and AST as compared to the INH plus RIF group.

Figure 1.

Effects of Dendrobine on the serum ALT and AST in liver injury mouse model induced by INH and RIF. Mice were administrated with 100 mg/kg INH and 100 mg/kg RIF for 14 days. Besides, mice were administered with different doses of dendrobine 2 h prior to the administration of INH and RIF. After the last administration for 16 h, mice were sacrificed and blood was collected. Serum ALT (a) and AST (b) were determined (n = 10). Data were represented as mean ± SD, ^# p > 0.05, ***p < 0.001. ALT: alanine aminotransferase; AST: aspartate aminotransferase; INH: isoniazid; RIF: rifampicin.

Effects of Dendrobine on liver injury induced by INH plus RIF

We then investigated the effects of Dendrobine on the liver injury induced by INH plus RIF via the evaluation of liver index and liver histopathology. The results showed that liver index was significantly increased in the INH plus RIF group (Figure 2(a)). However, treatment of Dendrobine (at 100 and 200 mg/kg) significantly decreased the liver index as compared to the INH plus RIF group.

Figure 2.

Effects of Dendrobine on liver injury induced by INH and RIF. After mice were sacrificed, the liver was collected. (a) The liver index of each group was measured. Besides, (b) liver histopathological examination was performed by using H&E staining (original magnification ×200) (n = 10). Data were represented as mean ± SD, ^# p > 0.05, ***p < 0.001. INF: isoniazid; RIF: rifampicin; H&E: hematoxylin and eosin.

Liver histopathological examinations showed that the structure of hepatocytes was normal in the control group (Figure 2(b)). However, we observed lipid infiltration and some balloon-like changes in the INH plus RIF group. The morphology of hepatocytes in the Dendrobine (50 mg/kg) was similar to the INH plus RIF group. However, treatment of Dendrobine (100 and 200 mg/kg) ameliorated these histopathological changes induced by INH plus RIF group.

Effects of Dendrobine on oxidative stress in the liver injury mouse model induced by INH plus RIF

To explore the effects of Dendrobine on the oxidative stress in liver, the levels of oxidative stress markers including GSH, SOD, and MDA were evaluated. The results showed that levels of GSH and SOD were significantly decreased in the INH plus RIF group as compared to the control group (Figure 3(a) and (b)). However, treatment of Dendrobine (at 100 and 200 mg/kg) significantly increased the levels of GSH and SOD in the liver as compared to the INH plus RIF group. Additionally, the levels of MDA were significantly increased in the INH plus RIF group as compared to the control group (Figure 3(c)). However, treatment of Dendrobine (at 100 and 200 mg/kg) significantly decreased the levels of GSH and SOD in liver as compared to the INH plus RIF group. These results suggested that treatment of Dendrobine (at 100 and 200 mg/kg) affected the oxidative stress in liver injury induced by INH plus RIF.

Figure 3.

Effects of Dendrobine on oxidative stress in the liver injury mouse model induced by INH and RIF. After the mice were sacrificed, the liver was collected and oxidative stress-related biomarkers including GSH (a), SOD (b), and MDA (c) were determined (n = 10). Data were represented as mean ± SD, ^# p > 0.05, ***p < 0.001. INF: isoniazid; RIF: rifampicin; GSH: glutathione; SOD: superoxide dismutase; MDA: malondialdehyde.

Effects of Dendrobine on the expressions of CYP1A2 and CYP2E1 in the liver injury mouse model induced by INH plus RIF

To explore the underlying mechanisms of Dendrobine on the oxidative stress, the levels of CYP1A2 and CYP2E1 were investigated. First, we determined the mRNA levels of CYP1A2 and CYP2E1. The results demonstrated that mRNA levels of CYP1A2 and CYP2E1 were significantly inhibited in the INH plus RIF group as compared to the control group (Figure 4(a) and (b)). However, treatment of Dendrobine (at 100 and 200 mg/kg) significantly increased the mRNA levels of CYP1A2 other than CYP2E1 (Figure 4(a)). To confirm the effects of Dendrobine on the CYP1A2, Western blot was applied. The results showed that treatment of Dendrobine (at 100 and 200 mg/kg) significantly increased the expressions of CYP1A2 (Figure 4(c) and (d)).

Figure 4.

Effects of Dendrobine on the expressions of CYP1A2 and CYP2E1 in the liver injury mouse model induced by INH and RIF. After the mice were sacrificed, the liver was collected. (a and b) qRT-PCR was performed to determine the mRNA levels of CYP1A2 and CYP2E1. Additionally, (c and d) Western blot was applied to determine the protein expressions of CYP1A2 in liver (n = 10). Data were represented as mean ± SD, ^# p > 0.05, **p < 0.01, ***p < 0.001. INF: isoniazid; RIF: rifampicin; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Dendrobine regulated the levels of mmu-miR-295-5p

To explore the target miRNAs that regulate CYP1A2, Targetscan was applied. The results showed that miRNAs including miR-7116-3p, miR-6951-3p, miR-6928-3p, miR-295-5p, miR-292a-5p, miR-290a-5p, miR-186-5p, miR-7118-5p, miR-183-3p, miR-143-3p, and miR-463-3p were possible targets of CYP1A2. Based on the prediction, qRT-PCR was applied to determine the levels of those miRNAs. Interestingly, miR-295-5p was significantly increased in the INH plus RIF group as compared to the control group, whereas treatment of Dendrobine (at 200 mg/kg) significantly decreased the levels of miR-295-5p (Figure 5).

Figure 5.

Dendrobine regulated the levels of mmu-miR-295-5p. Firstly, miRNAs were predicted by the online tool Targetscan (http://www.targetscan.org). After that, qRT-PCR was performed to determine the expressions of miRNAs in the liver (n = 5). Data were represented as mean ± SD, ***p < 0.001. qRT-PCR: quantitative reverse transcription polymerase chain reaction.

mmu-miR-295-5p targeted CYP1A2 in the AML12 cells

To confirm the interactions between mmu-miR-295-5p targeted CYP1A2, luciferase reporter assay was applied. The results showed that luciferase activities in the cells that were co-transfected plasmids containing a sequence of CYP1A2 3′UTR WT with mmu-miR-295-5p were dramatically decreased (Figure 6(a)). Interestingly, we did not observe the change of luciferase activities in the cells that were co-transfected plasmids containing sequence of CYP1A2 3′UTR MUT with mmu-miR-295-5p as compared to cells co-transfected CYP1A2 3′UTR MUT with the scramble. In addition, we also observed that miR-295-5p overexpression significantly downregulated CYP1A2, while downregulation of miR-295-5p did not upregulate CYP1A2 (Figure 6(b)). Due to low basal level of miR-295-5p in the cells, we did not observe the significant change of CYP1A2 as compared to the scramble group. Overall, these results supported that mmu-miR-295-5p targeted CYP1A2 in the AML12 cells.

Figure 6.

mmu-miR-295-5p targeted CYP1A2 in the AML12 cells. (a) Dual-luciferase reporter gene assay was performed to determine the interactions between mmu-miR-295-5p and CYP1A2 3′UTR in AML12 cells. (b) Directly overexpressed mmu-miR-29505p could decrease the mRNA level of CYP1A2.

Discussion

Tuberculosis is an infectious disease caused by the bacteria called Mycobacterium tuberculosis.²¹ There are approximately 10 million tuberculosis patients in 2018. INH and RIF are the first-line medications in tuberculosis therapy.^4,6 However, their uses cause hepatotoxicity including liver injury and liver failure in some patients.⁵ INH is metabolized into hydrazine by CYP450, which induces liver steatosis. When INH is combined with RIF, RIF is converted into desacetyl RIF by CYP450 and thereby promoting hepatotoxicity.^3,5 Thus, it is important to discover drug candidates to reverse liver toxicities induced by INH and/or RIF.

In the present study, a mouse model of liver injury was firstly established by coadministration 100 mg/kg INH with 100 mg/kg RIF. We observed that serum AST and ALT were significantly increased along with lipid infiltration and some balloon-like changes in liver. These results are also consistent with a previous study, in which coadministration of 75 mg/kg INH with 150 mg/kg RIF induces (1) evaluation of plasma ALT, (2) accumulation of liver lipid, (3) and hepatocytes apoptosis.²²

Our results demonstrated that the administration of Dendrobine (100 and 200 mg/kg) significantly decreased serum ALT and AST. Both ALT and AST are commonly used markers for the evaluation of liver injury.^23,24 AST is responsible for the transferase of the amino group and is usually elevated due to liver dysfunction including liver cell infection or necrosis.²⁴ ALT is a transaminase enzyme widely used in the clinical examination for liver function tests. The increase of blood ALT is associated with a series of liver dysfunction problems including liver injury and infection of viral hepatitis.²³ In the present study, administration of INH with RIF significantly increased serum ALT and AST, whereas treatment of Dendrobine (100 and 200 mg/kg) significantly decreased serum ALT and AST, indicating Dendrobine possessed liver protective effects. Consistent with these results, we also observed that Dendrobine (100 and 200 mg/kg) ameliorated liver histopathological changes induced by INH and RIF.

Oxidative stress has been implicated to play a crucial role in liver injury.^25,26 In the present study, we evaluated the effects of Dendrobine on the oxidative stress markers including GSH, SOD, and MDA. MDA is an indicator to evaluate the amount of polyunsaturated membrane lipid that has been oxidized.²⁷ SOD is an enzyme for the clearance of oxidative radicals.²⁷ We observed liver MDA was significantly increased, whereas liver SOD was decreased in the INH and RIF group. Interestingly, Dendrobine (100 and 200 mg/kg) inhibited liver MDA and promoted liver SOD. Additionally, we also determined liver an antioxidant biomarker GSH. The depletion of GSH induces the production of reactive oxygen species.²⁷ Dendrobine (100 and 200 mg/kg) significantly increased liver GSH as compared to the INH and RIF group. Furthermore, we explored the underlying mechanisms of Dendrobine on the regulation of liver injury. CYP1A2 and CYP2E1 are two key enzymes that are evaluated due to the administration of INH and RIF.^4,5 Interestingly, we observed Dendrobine (100 and 200 mg/kg) significantly increased the levels of CYP1A2. These results supported that Dendrobine affected oxidative stress biomarkers in the liver by the regulation of CYP1A2.

In the present study, we identified miR-295-5p as a target of Dendrobine. Luciferase reporter assay supported that mmu-miR-295-5p targeted CYP1A2 by direct interactions. A recent study has identified that miR-295-5p is involved in the nonalcoholic fatty liver disease and is associated with triglyceride accumulation.¹⁸ However, the functions of mmu-miR-295-5p are still unknown in the liver injury. We identified an increase of mmu-miR-295-5p in the liver injury, indicating that mmu-miR-295-5p might be a biomarker associated with liver injury. However, investigations on its underlying mechanisms are warranted in further study.

In 2019, Li and colleagues have reported that D. nobile Lindl. alkaloids protect acute liver injury from carbon tetrachloride by the induction of nuclear factor erythroid 2-related factor 2 signaling pathway.²⁸ However, this study does not identify the specific alkaloid with liver protective effect as the alkaloids that used in the study contain Dendrobine, Dendrobine-N-oxide, Nobilonine, Dendroxine, 6-Hydroxy-nobilonine, and 13-Hydroxy-14-oxodendrobine.²⁸ In the present study, for the first time, we identified Dendrobine as a potential alkaloid for the treatment of INH- and RIF-induced liver injury. Moreover, this study also revealed the effects of Dendrobine on INH- and RIF-induced in part by the regulation of miR-295-5p-mediated CYP1A2 expression. These preliminary results suggested that Dendrobine might be a drug candidate for the treatment of liver injury. However, the underlying mechanisms of Dendrobine on INH- and RIF-induced liver injury are still not fully understood. Besides, clinical settings are warranted to support the use of Dendrobine on the treatment of INH- and RIF-induced liver injury.

Footnotes

Authors’ note

R Ci and K Zhang contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

K Zhang

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