# Antioxidant and Anti-Inflammatory Activities of Coenzyme-Q10 and Piperine against Cyclophosphamide-Induced Cytotoxicity in HuH-7 Cells

Authors: Norah S. AL-Johani; Mohammed Al-Zharani; Nada H. Aljarba; Norah M. Alhoshani; Nora Alkeraishan; Saad Alkahtani
Journal: BioMed Research International
 (2022)

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## Abstract

Cyclophosphamide (CP) alkylates DNA and RNA produce crosslinks that cause gene expression and protein synthesis inhibition to exert its anticancer effect. However, adverse effects of CP have restricted the CP application in cancer treatment. We investigate coenzyme-Q10 (Q10) and piperine (P) protective role on CP oxidant and inflammatory effect. HuH-7 cells were exposed to varying concentrations and combinations of Q10, P, and CP and evaluated intracellular ROS generation as well as inflammatory responses upon exposure. Our results showed Q10 and/or P suppressed both basal and CP-induced ROS generation without upsetting the balance in activities of SOD, catalase, and GSH levels. Analysis of proinflammatory cytokine gene expression showed that CP treatment alone only induced expression ofIL-6β. However, coexposure of the cells to both Q10 and CP caused significant suppression of basal Cox-2 and TNF-α gene expression, while coexposure of the cells to CP and P with Co-Q10 suppressed basal IL-1β gene expression. Q10 also suppressed CP-induced expression of Cox-1. P and CP suppressed basal expression of IL-6β and IL-12β, while P and Q10 suppressed CP-induced IL1-α gene expression. Taken together, both Q10 and P seem to be inhibiting NFκβ pathway to suppress CP-mediated inflammation. In conclusion, Q10 and/or P induced suppression of ROS generation mediated by CP and also suppressed CP-induced inflammation by inhibiting expression of specific inflammatory cytokine.


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## Body

## 1. Introduction

Several chemotherapeutic drugs used to treat different types of cancer diseases. Some of those drugs have been failed due to adverse effect exhibited by the drugs preventing their continued application in treating cancer patients. One such compound which is widely restricted is cyclophosphamide (CP). CP is an anticancer drug used in treatment of various solid tumors such as in the breast, lung, and prostate cancers, in addition to blood disorders like leukemia and lymphoma [1–4]. Unfortunately, adverse effects such as hepatotoxicity, nephrotoxicity, cardiotoxicity, and severe immunotoxicity have restricted the use of CP in cancer treatment [5].CP is an alkylating agent metabolized by cytochrome p450 (CYP450) liver enzyme to phosphoramide and acrolein, in which alkylates DNA and RNA produce crosslinks that inhibit gene expression and protein synthesis [6]. Furthermore, CP metabolism results in generation of active carbonium ions, an electrophile that attack electron rich nucleic acids in DNA, RNA, and proteins causing DNA damage and producing reactive oxygen species (ROS) [7]. The ROS generated due to administration of CP further result in DNA damage and perturbation in the oxidant-antioxidant balance in the cell, negatively affecting cellular homeostasis even in normal healthy cells.One downstream effect of ROS generation is induction of inflammation. ROS generation induces activation of different transcription factors like NFκβ, which regulates expression of proinflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) which subsequently result in activating cycloxegenase-2 (COX-2) [8]. Interestingly, there are studies on chemotherapeutic agents such as CP inducing inflammation via ROS-induced activation of NFκβ [9]. In addition, NFκβ activation may also result in activation of additional transcription factors such as STAT-3 which in turn activates IL-6 expression creating sustained inflammatory response loop [10]. Girard et al. [11] showed in rat model that CP induced bladder inflammation through activation of JAK/STAT pathway involving expression of IL-6 and IL-6Rα. Moschella et al. [12] also demonstrated in a mouse model that CP treatment resulted in expression of gene and protein belonging to IL-1 family members such as IL-1β, IL-18, and interferon γ (IFN-γ).In order to prevent or minimize the adverse effects of CP and its metabolite to improve it for therapy in cancer is to employ less toxic drugs. Natural antioxidant products are explored for therapeutic purposes because such perceived safety against normal healthy cells. Piperine (P) is an alkaloid that is found in black pepper (Piper nigrum L.). P is a pharmacoactive agent known to possess hepatoprotective, antioxidant, and immunomodulatory effects [13]. Similarly, coenzyme Q10 (Q10) is a naturally occurring compound in mammalian cells with repeating isoprene units that has been investigated for alleviating the toxicity of different compounds due to its antioxidant properties [14]. For instance, Q10 has been used to suppress cisplatin-mediated oxidative stress which induces inflammation, necrosis, and apoptosis in renal tissue via its antioxidant activities [15]. Similarly, Q10 has been used to alleviate toxicity of anthracycline, tamoxifen, and doxorubicin [16–18]. Here, we explored P and Q10 as protective agents against inflammation and oxidative stress induced by CP. We investigated CP cytotoxic effect alone and with Q10 and P in HuH-7 cell line and evaluate the oxidative stress, antioxidant levels, and inflammatory responses in HuH-7 cell line.


## 2. Methodology

### 2.1. Preparation of the Treatments

CP (MedChemExpress LLC, USA), P, and Q10 (BioPiperine®USA) were dissolved in DMSO to make 1 mg/1 ml.


### 2.2. MTT Assay

HuH-7 (ATCC, USA) were cultured in 70% DMEM supplemented with 30% FBS and 1% penicillin-streptomycin and incubated at 5% CO2 and 37°C. Cell viability and proliferation were both determined using an MTT assay (Mosmann, 1983). Cells were cultured in 24-well plate after which they were incubated at above conditions for 24 h. The cells were exposed to predetermined concentrations of P, CP, Q10 alone, or combination of P and/or Q10 with CP for 48 h. MTT solution (100 μl) was added, and cells were returned to incubator for 4 h. Medium was aspirated from the culture plate, and formed crystals were dispersed in isopropanol (1 ml/well) in 0.04 HCl followed by shaking at RT for 12 minutes to ensure complete solubilization. Afterwards, 100 μl of the media from each well was added into a 96-well plate for absorbance measurement at 540 nm (multimode Microplate Reader-Gen5™, BioTek Cytation 5™, USA).


### 2.3. Lactate Dehydrogenase Assay (LDH Assay)

Cell viability and membrane integrity were assessed by performing LDH assay kit (MAK066 Sigma-Aldrich, St Louis, USA). Briefly, the cells were seeded as above 100μl of medium, then incubated under above conditions for 24 h. The media was aspirated, discarded, and then, replaced with 200 μl of new medium for each well. Cells were then exposed to 10 μg/ml of CP, 12 μg/ml P, 10 μg/ml Q10, 12 μg/ml of P + Q10, and combination of CP + P, CP + Q10, and CP+ P+ Q10 as in the individual concentrations for 48 h. Then, the supernatant (50 μl) was transferred to new 96-well plate followed by addition of 50 μl of the Master Reaction Mix to each well. The cells were mixed and incubated on a horizontal shaker for 3 min in the dark. The OD was measured at 450 nm using the spectrophotometer. The plate was reincubated, and OD was measured twice every 5 mins.


### 2.4. Oxidative Stress

#### 2.4.1. Production of ROS

ROS production was evaluated by H2DCF-DA (Dikalov et al., 2007). ROS was assayed using the Cayman-ROS Detection Cell-Based Assay Kit following manufacturer’s instruction. Briefly, the HuH-7 cells were seeded in black 96-well plate of 5×104/well for 24 h under above incubation conditions. Cells were exposed to each treatment for 48 h as above, and then, the media was replaced with 100 μl of diluted H2DCF-DA dye solution, which was prepared by adding 1 μl of H2DCF-DA dye to 99 μl of MEME in the dark; the plates were returned to the incubator for 60 min. Absorbance analysis was done at excitation wavelengths of 480 and emission 530 nm in a microplate reader. To monitor the intracellular ROS under microscope, cells were seeded in 6-well plate and incubated for 24 h. After that cell were exposed as above for 48 h, then the medium was replaced with 1 ml diluted H2DCF-DA solution in the dark 1 h. The cells were washed in prewarmed PBS for 3 times. Fluorescent images were taken by DMLB fluorescence microscope (Leica, Germany) and analyzed using the ImageJ software (version 1.51, NIH, Bethesda, MD, USA).


#### 2.4.2. Catalase Activity

The assessment of CAT activity was achieved by catalase colorimetric Kit, K773-100, BioVision. Briefly, the HuH-7 cells seeded in 25 ml flask of1×106 cells/ml culture media were incubated for 24 h. Treatments were applied as before. After incubation, cells were washed, and then, 1 ml cold assay buffer was added. Immediately, cells were scraped and vortexed for 2 min, followed by centrifuging at 12000 rpm at 4°C for 7 min. After this, samples were added/well, and the total volume was adjusted to 78 μl with assay buffer. To stop the reaction, 10 μl of stop solution was added, and then, 12 μl of fresh (1 mM H2O2) was added to start the reaction. Cells were incubated for 30 min at 25°C, and 10 μl of stop solution was added to each sample well. The developer mix (50 μl) was added to each sample, mixed well, and incubated for 10 min at 25°C. OD was then measured at 570 nm.


#### 2.4.3. Superoxide Dismutase (SOD)

The assessment of SOD activity was conducted by SOD kit (Cayman Chemical, Michigan USA). The HuH-7 cells were seeded in 25 ml flask of1×106 cells/well for 24 h. Treatments were done as above h. Cells were washed, and 1 ml cold SOD lysis buffer (pH 7.2) was added. Cells were collected and sonicated for 2 min, then centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant was transferred into new Eppendorf tubes, and 200 μl of radical detector was added, followed by adding 10 μl of each sample (cell lysates), and then, 20 μl of diluted xanthine oxidase was added. The cells were incubated at RT for 10 min. OD was measured at 460 nm (Synergy-H1; BioTek).


#### 2.4.4. Glutathione (GSH)

Evaluation of total glutathione (GSH) content was analysed using Glutathione assay kit (Cayman Chemical, Michigan USA). Briefly, the HuH-7 cells were cultivated in 25 ml flask for 24 h. After treatment 1 ml of cold buffer (0.4 M 2-(N-morpholino) ethanesulfonic acid, 0.1 M phosphate, and 2 mM EDTA,pH=6) at 4° C was added to each flask. Cells were collected and sonicated for 2 min after which they were 2 centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant and 50 μl of sample (cell lysate from treated and control cells) were added to each well. A 150 μl of freshly prepared cocktail assay was added and mixed well by pipetting; plate was in the dark on an orbital shaker. OD was recorded at 405 nm (Synergy-H1; BioTek).


### 2.5. Gene Expression

#### 2.5.1. RNA Extraction

Briefly, HuH-7 cells were cultured and exposed as before. After incubation, the cells were washed with cold PBS, and 2 ml of Trizol was added to each flask on ice for 5 min. 1 ml of cells was transferred into Eppendorf tubes (1 ml/tube) after which 200μl of cold chloroform was added. Tubes were mixed and then centrifuged at 15000 rpm for 10 min at 4°C. Then, 500 μl of supernatant was added followed by addition of 500 μl iso-propanol alcohol to each tube, gently mixed, and incubated for 10 min on ice followed by centrifuged at 12000 rpm for 15 min at 4°C. Supernatant was discarded; 1 ml of cold absolute ethanol was added and gently mixed for 15 sec. Tubes were centrifuged at 10000 rpm for 5 min and 4°C, and the step was repeated. Supernatant was discarded, and tubes were allowed to dry for 15 min, and 30 μl of DEPC water was added. Concentration and purity of RNA were measured by Nanodrop 8000 (Thermo Fisher Scientific, USA).


#### 2.5.2. Complementary DNA (cDNA) Synthesis

cDNA synthesis kit GoScript™ Reverse Transcriptase (CAT No A5003, Promega, USA) was carried out according to manufacturer instructions. Briefly, 0.5μl Oligo (dT) and 0.5 μl random hexamer primer were added to a PCR tube. Total RNA and nuclease-free water were added up to 5 μl total volume. PCR tubes were placed in the thermal cycler (TECHNE, UK) at 70°C for 5 min. Afterwards, 15 μl of mater-mix was added to each sample and placed in thermal cycler. After cycle completion, 180 μl of nuclease-free water was added to each cDNA sample (total volume of each sample was 200 μl).


#### 2.5.3. Quantification of mRNA Expression

Quantitative analysis of mRNA expression was performed by RT-PCR through PCR amplification (cDNA) in CFX96™ real-time system (BIO-RAD, USA) using KAPA SYBER FAST Universal qPCR Kit (Cat No. KK4600, KAPA BIOSYSTEM, USA). The RT-PCR data analysis was done using the relative gene expression (i.e.,ΔΔCt) method, as described previously (Livak and Schmittgen, 2001).


### 2.6. Statistical Analysis

The SPSS software (ver.22; SPSS Inc., Chicago, IL, USA) was used to analyze the obtained data. Data were examined by using two-way ANOVA, followed by a post hoc LSD (least significant difference) test, and results were presented asaverage±SE. p<0.05 were considered statistically significant.


## 2.1. Preparation of the Treatments

CP (MedChemExpress LLC, USA), P, and Q10 (BioPiperine®USA) were dissolved in DMSO to make 1 mg/1 ml.


## 2.2. MTT Assay

HuH-7 (ATCC, USA) were cultured in 70% DMEM supplemented with 30% FBS and 1% penicillin-streptomycin and incubated at 5% CO2 and 37°C. Cell viability and proliferation were both determined using an MTT assay (Mosmann, 1983). Cells were cultured in 24-well plate after which they were incubated at above conditions for 24 h. The cells were exposed to predetermined concentrations of P, CP, Q10 alone, or combination of P and/or Q10 with CP for 48 h. MTT solution (100 μl) was added, and cells were returned to incubator for 4 h. Medium was aspirated from the culture plate, and formed crystals were dispersed in isopropanol (1 ml/well) in 0.04 HCl followed by shaking at RT for 12 minutes to ensure complete solubilization. Afterwards, 100 μl of the media from each well was added into a 96-well plate for absorbance measurement at 540 nm (multimode Microplate Reader-Gen5™, BioTek Cytation 5™, USA).


## 2.3. Lactate Dehydrogenase Assay (LDH Assay)

Cell viability and membrane integrity were assessed by performing LDH assay kit (MAK066 Sigma-Aldrich, St Louis, USA). Briefly, the cells were seeded as above 100μl of medium, then incubated under above conditions for 24 h. The media was aspirated, discarded, and then, replaced with 200 μl of new medium for each well. Cells were then exposed to 10 μg/ml of CP, 12 μg/ml P, 10 μg/ml Q10, 12 μg/ml of P + Q10, and combination of CP + P, CP + Q10, and CP+ P+ Q10 as in the individual concentrations for 48 h. Then, the supernatant (50 μl) was transferred to new 96-well plate followed by addition of 50 μl of the Master Reaction Mix to each well. The cells were mixed and incubated on a horizontal shaker for 3 min in the dark. The OD was measured at 450 nm using the spectrophotometer. The plate was reincubated, and OD was measured twice every 5 mins.


## 2.4. Oxidative Stress

### 2.4.1. Production of ROS

ROS production was evaluated by H2DCF-DA (Dikalov et al., 2007). ROS was assayed using the Cayman-ROS Detection Cell-Based Assay Kit following manufacturer’s instruction. Briefly, the HuH-7 cells were seeded in black 96-well plate of 5×104/well for 24 h under above incubation conditions. Cells were exposed to each treatment for 48 h as above, and then, the media was replaced with 100 μl of diluted H2DCF-DA dye solution, which was prepared by adding 1 μl of H2DCF-DA dye to 99 μl of MEME in the dark; the plates were returned to the incubator for 60 min. Absorbance analysis was done at excitation wavelengths of 480 and emission 530 nm in a microplate reader. To monitor the intracellular ROS under microscope, cells were seeded in 6-well plate and incubated for 24 h. After that cell were exposed as above for 48 h, then the medium was replaced with 1 ml diluted H2DCF-DA solution in the dark 1 h. The cells were washed in prewarmed PBS for 3 times. Fluorescent images were taken by DMLB fluorescence microscope (Leica, Germany) and analyzed using the ImageJ software (version 1.51, NIH, Bethesda, MD, USA).


### 2.4.2. Catalase Activity

The assessment of CAT activity was achieved by catalase colorimetric Kit, K773-100, BioVision. Briefly, the HuH-7 cells seeded in 25 ml flask of1×106 cells/ml culture media were incubated for 24 h. Treatments were applied as before. After incubation, cells were washed, and then, 1 ml cold assay buffer was added. Immediately, cells were scraped and vortexed for 2 min, followed by centrifuging at 12000 rpm at 4°C for 7 min. After this, samples were added/well, and the total volume was adjusted to 78 μl with assay buffer. To stop the reaction, 10 μl of stop solution was added, and then, 12 μl of fresh (1 mM H2O2) was added to start the reaction. Cells were incubated for 30 min at 25°C, and 10 μl of stop solution was added to each sample well. The developer mix (50 μl) was added to each sample, mixed well, and incubated for 10 min at 25°C. OD was then measured at 570 nm.


### 2.4.3. Superoxide Dismutase (SOD)

The assessment of SOD activity was conducted by SOD kit (Cayman Chemical, Michigan USA). The HuH-7 cells were seeded in 25 ml flask of1×106 cells/well for 24 h. Treatments were done as above h. Cells were washed, and 1 ml cold SOD lysis buffer (pH 7.2) was added. Cells were collected and sonicated for 2 min, then centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant was transferred into new Eppendorf tubes, and 200 μl of radical detector was added, followed by adding 10 μl of each sample (cell lysates), and then, 20 μl of diluted xanthine oxidase was added. The cells were incubated at RT for 10 min. OD was measured at 460 nm (Synergy-H1; BioTek).


### 2.4.4. Glutathione (GSH)

Evaluation of total glutathione (GSH) content was analysed using Glutathione assay kit (Cayman Chemical, Michigan USA). Briefly, the HuH-7 cells were cultivated in 25 ml flask for 24 h. After treatment 1 ml of cold buffer (0.4 M 2-(N-morpholino) ethanesulfonic acid, 0.1 M phosphate, and 2 mM EDTA,pH=6) at 4° C was added to each flask. Cells were collected and sonicated for 2 min after which they were 2 centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant and 50 μl of sample (cell lysate from treated and control cells) were added to each well. A 150 μl of freshly prepared cocktail assay was added and mixed well by pipetting; plate was in the dark on an orbital shaker. OD was recorded at 405 nm (Synergy-H1; BioTek).


## 2.4.1. Production of ROS

ROS production was evaluated by H2DCF-DA (Dikalov et al., 2007). ROS was assayed using the Cayman-ROS Detection Cell-Based Assay Kit following manufacturer’s instruction. Briefly, the HuH-7 cells were seeded in black 96-well plate of 5×104/well for 24 h under above incubation conditions. Cells were exposed to each treatment for 48 h as above, and then, the media was replaced with 100 μl of diluted H2DCF-DA dye solution, which was prepared by adding 1 μl of H2DCF-DA dye to 99 μl of MEME in the dark; the plates were returned to the incubator for 60 min. Absorbance analysis was done at excitation wavelengths of 480 and emission 530 nm in a microplate reader. To monitor the intracellular ROS under microscope, cells were seeded in 6-well plate and incubated for 24 h. After that cell were exposed as above for 48 h, then the medium was replaced with 1 ml diluted H2DCF-DA solution in the dark 1 h. The cells were washed in prewarmed PBS for 3 times. Fluorescent images were taken by DMLB fluorescence microscope (Leica, Germany) and analyzed using the ImageJ software (version 1.51, NIH, Bethesda, MD, USA).


## 2.4.2. Catalase Activity

The assessment of CAT activity was achieved by catalase colorimetric Kit, K773-100, BioVision. Briefly, the HuH-7 cells seeded in 25 ml flask of1×106 cells/ml culture media were incubated for 24 h. Treatments were applied as before. After incubation, cells were washed, and then, 1 ml cold assay buffer was added. Immediately, cells were scraped and vortexed for 2 min, followed by centrifuging at 12000 rpm at 4°C for 7 min. After this, samples were added/well, and the total volume was adjusted to 78 μl with assay buffer. To stop the reaction, 10 μl of stop solution was added, and then, 12 μl of fresh (1 mM H2O2) was added to start the reaction. Cells were incubated for 30 min at 25°C, and 10 μl of stop solution was added to each sample well. The developer mix (50 μl) was added to each sample, mixed well, and incubated for 10 min at 25°C. OD was then measured at 570 nm.


## 2.4.3. Superoxide Dismutase (SOD)

The assessment of SOD activity was conducted by SOD kit (Cayman Chemical, Michigan USA). The HuH-7 cells were seeded in 25 ml flask of1×106 cells/well for 24 h. Treatments were done as above h. Cells were washed, and 1 ml cold SOD lysis buffer (pH 7.2) was added. Cells were collected and sonicated for 2 min, then centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant was transferred into new Eppendorf tubes, and 200 μl of radical detector was added, followed by adding 10 μl of each sample (cell lysates), and then, 20 μl of diluted xanthine oxidase was added. The cells were incubated at RT for 10 min. OD was measured at 460 nm (Synergy-H1; BioTek).


## 2.4.4. Glutathione (GSH)

Evaluation of total glutathione (GSH) content was analysed using Glutathione assay kit (Cayman Chemical, Michigan USA). Briefly, the HuH-7 cells were cultivated in 25 ml flask for 24 h. After treatment 1 ml of cold buffer (0.4 M 2-(N-morpholino) ethanesulfonic acid, 0.1 M phosphate, and 2 mM EDTA,pH=6) at 4° C was added to each flask. Cells were collected and sonicated for 2 min after which they were 2 centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant and 50 μl of sample (cell lysate from treated and control cells) were added to each well. A 150 μl of freshly prepared cocktail assay was added and mixed well by pipetting; plate was in the dark on an orbital shaker. OD was recorded at 405 nm (Synergy-H1; BioTek).


## 2.5. Gene Expression

### 2.5.1. RNA Extraction

Briefly, HuH-7 cells were cultured and exposed as before. After incubation, the cells were washed with cold PBS, and 2 ml of Trizol was added to each flask on ice for 5 min. 1 ml of cells was transferred into Eppendorf tubes (1 ml/tube) after which 200μl of cold chloroform was added. Tubes were mixed and then centrifuged at 15000 rpm for 10 min at 4°C. Then, 500 μl of supernatant was added followed by addition of 500 μl iso-propanol alcohol to each tube, gently mixed, and incubated for 10 min on ice followed by centrifuged at 12000 rpm for 15 min at 4°C. Supernatant was discarded; 1 ml of cold absolute ethanol was added and gently mixed for 15 sec. Tubes were centrifuged at 10000 rpm for 5 min and 4°C, and the step was repeated. Supernatant was discarded, and tubes were allowed to dry for 15 min, and 30 μl of DEPC water was added. Concentration and purity of RNA were measured by Nanodrop 8000 (Thermo Fisher Scientific, USA).


### 2.5.2. Complementary DNA (cDNA) Synthesis

cDNA synthesis kit GoScript™ Reverse Transcriptase (CAT No A5003, Promega, USA) was carried out according to manufacturer instructions. Briefly, 0.5μl Oligo (dT) and 0.5 μl random hexamer primer were added to a PCR tube. Total RNA and nuclease-free water were added up to 5 μl total volume. PCR tubes were placed in the thermal cycler (TECHNE, UK) at 70°C for 5 min. Afterwards, 15 μl of mater-mix was added to each sample and placed in thermal cycler. After cycle completion, 180 μl of nuclease-free water was added to each cDNA sample (total volume of each sample was 200 μl).


### 2.5.3. Quantification of mRNA Expression

Quantitative analysis of mRNA expression was performed by RT-PCR through PCR amplification (cDNA) in CFX96™ real-time system (BIO-RAD, USA) using KAPA SYBER FAST Universal qPCR Kit (Cat No. KK4600, KAPA BIOSYSTEM, USA). The RT-PCR data analysis was done using the relative gene expression (i.e.,ΔΔCt) method, as described previously (Livak and Schmittgen, 2001).


## 2.5.1. RNA Extraction

Briefly, HuH-7 cells were cultured and exposed as before. After incubation, the cells were washed with cold PBS, and 2 ml of Trizol was added to each flask on ice for 5 min. 1 ml of cells was transferred into Eppendorf tubes (1 ml/tube) after which 200μl of cold chloroform was added. Tubes were mixed and then centrifuged at 15000 rpm for 10 min at 4°C. Then, 500 μl of supernatant was added followed by addition of 500 μl iso-propanol alcohol to each tube, gently mixed, and incubated for 10 min on ice followed by centrifuged at 12000 rpm for 15 min at 4°C. Supernatant was discarded; 1 ml of cold absolute ethanol was added and gently mixed for 15 sec. Tubes were centrifuged at 10000 rpm for 5 min and 4°C, and the step was repeated. Supernatant was discarded, and tubes were allowed to dry for 15 min, and 30 μl of DEPC water was added. Concentration and purity of RNA were measured by Nanodrop 8000 (Thermo Fisher Scientific, USA).


## 2.5.2. Complementary DNA (cDNA) Synthesis

cDNA synthesis kit GoScript™ Reverse Transcriptase (CAT No A5003, Promega, USA) was carried out according to manufacturer instructions. Briefly, 0.5μl Oligo (dT) and 0.5 μl random hexamer primer were added to a PCR tube. Total RNA and nuclease-free water were added up to 5 μl total volume. PCR tubes were placed in the thermal cycler (TECHNE, UK) at 70°C for 5 min. Afterwards, 15 μl of mater-mix was added to each sample and placed in thermal cycler. After cycle completion, 180 μl of nuclease-free water was added to each cDNA sample (total volume of each sample was 200 μl).


## 2.5.3. Quantification of mRNA Expression

Quantitative analysis of mRNA expression was performed by RT-PCR through PCR amplification (cDNA) in CFX96™ real-time system (BIO-RAD, USA) using KAPA SYBER FAST Universal qPCR Kit (Cat No. KK4600, KAPA BIOSYSTEM, USA). The RT-PCR data analysis was done using the relative gene expression (i.e.,ΔΔCt) method, as described previously (Livak and Schmittgen, 2001).


## 2.6. Statistical Analysis

The SPSS software (ver.22; SPSS Inc., Chicago, IL, USA) was used to analyze the obtained data. Data were examined by using two-way ANOVA, followed by a post hoc LSD (least significant difference) test, and results were presented asaverage±SE. p<0.05 were considered statistically significant.


## 3. Results

### 3.1. P and Q10 Exhibit Lower Cytotoxicity vs. CP

MTT assay was performed to determine the cytotoxicity of CP and impact of P and Q10 on CP’s cytotoxicity, postexposure to the compounds. We observed dose-dependent decrease of cell survival for all the compounds. Both P and Q10 had similar impact on cell viability at 5μg/ml compared to unexposed cells while CP significantly induced cell viability at 5 μg/ml compared to the control unexposed cells. Similarly, we found that coexposure of the HuH-7 resulted in less cytotoxic effect compared to CP (Table 1).Table 1
Cell viability of HuH-7 cells after treatment with CP, PIP, CoQ-10, and PIP+CoQ-10 for 48 hrs as evaluated by MTT assay. Each value represents themean±SE (n=3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) compared with control.
TreatmentsConcentrations (μg/ml)AverageViability (%)IC50 (μg/ml)Control—0.557±0.001100CP50.373±0.00367.05∗∗10100.273±0.00349.1∗∗∗150.175±0.00431.5∗∗200.138±0.00624.8∗∗∗250.093±0.00216.6∗∗∗PIP50.523±0.1293.916100.382±0.0768.6150.275±0.00749.4∗∗200.231±0.0241.5∗∗250.186±0.0233.4∗Q1050.44±0.00178.915100.368±0.0166∗150.277±0.0249.7∗200.206±0.0137∗∗250.161±0.0228.9∗PIP+Q1050.42±0.0775.418100.387±0.0469.4150.338±0.0560.7200.258±0.0546.4250.231±0.0541.4


### 3.2. P and Q10 Enhance Permeabilisation of HuH-7 Cell Membrane

Findings from LDH assay indicated a significant loss of membrane integrity after exposure of the cells to all investigated compounds when compared to untreated cells after 48 h (Figure1). However, combination of CP+P or CP+Q10 or CP with both P and Q10 significantly increased permeability of the cell membrane compared to CP. This indicates P/Q10 seems to enhance cell membrane permeabilisation effect of CP alone.Figure 1
Effect of CP, PIP, and CoQ-10 on LDH % in HuH-7 cell lines when treated for 48 h (∗p<0.05, ∗∗∗p<0.001) compared to the control group (#p<0.05, ##p<0.01) compared with CP.


### 3.3. Induction of (ROS)

A significant increase in ROS level posttreatment with CP compared with control unexposed cells (p<0.05). Contrastingly, single exposure to either of P or Q10 alone or combination of both compounds resulted in significant suppression of ROS generation compared to control cells (p<0.05). Furthermore, coexposure of the cells to either of CP+P, CP+Q10, and CP+P+ Q10 significantly suppressed ROS generation compared to exposure to CP alone (Figures 2(a) and 2(b)).Figure 2
(a) The production of ROS in HuH-7 cells as stained with fluorescence dye (DCFH-DA) after treatments. Bar: 400μm. (b) Induction of ROS levels in HuH-7 after treatment with CP, PIP, and CoQ-10 for 48 h. Each value represents the mean±SE (n=3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) compared with control (#p<0.05, ##p<0.01, ###p<0.001) compared with CP.
(a)(b)


### 3.4. Catalase (CAT) Activity

Based on the suppressed ROS generation observed above, we proceeded to assess catalase activity in the cell postexposure to the compounds. Our findings showed significant reduction in CAT activity posttreatment with P, CP+Q10, and CP+P+Q10 was significantly reduced after 48 h (∗p<0.05, ∗∗p<0.01) (Figure 3).Figure 3
CAT levels in HuH-7 after treatment with CP, PIP, Q-10, and in combination of CP with each compound for 48 hr. Data present represents themean±SE (n=3) (∗p<0.05, ∗∗p<0.01) compared with untreated control.


### 3.5. Superoxide Dismutase (SOD)

This enzyme catalyzes the conversion of superoxide radical into less-toxic hydrogen peroxides to help healthy cells actively fight oxidative stress. After treatment, a significant drop in activity of SOD due to CP exposure was observed compared to the control cells (∗∗p<0.01). In addition, reduction in SOD activity was observed after treatment with other investigated compounds compared to control although these were not significant. Similarly, a nonsignificant increase in SOD activity was observed after treatment with CP+P, CP+Q10, and CP with both P and Q10 compared to CP alone (Figure 4).Figure 4
SOD levels in HuH-7 after treatment with CP, PIP, CoQ-10, and in combination of CP with each compound for 48 hr. Data present represents themean±SE (n=3) (∗p<0.05) compared with untreated control.


### 3.6. Glutathione (GSH)

Glutathione (GSH) plays a crucial function in removal of many reactive species. HuH-7 cells were exposed to CP, P, Q10, and in combination with CP. Our result as shown in (Figure5) indicated nonsignificant reduction in GSH level when exposed to CP, P, and Q10 but there was significant decrease in GSH level after treatment with combination of P+Q10 and combination of CP+P and CP+P+ Q10 compared to CP.Figure 5
GSH levels in HuH-7 after treatment with CP, PIP, Q-10, and in combination of CP with each compound for 48 hr. Data present represents themean±SE (n=3) (∗∗p<0.01) compared with untreated control (#p<0.05, ##p<0.01) compared with CP.


### 3.7. Gene Expression

#### 3.7.1. Inflammatory Marker Genes at Transcriptional Level

Gene expression ofCox-1 was found higher in cells exposed to CP alone. However, coexposure of the cells to CP and Q10 resulted in significant reduction in Cox-1 gene expression (#p<0.05). We found that coexposure of the cells to CP and either of P or P+Q10 as well as single exposure to P alone resulted in considerable reduction of Cox-1 expression (Figure 6). Assessment of Cox-2 expression produced a contrasting finding to that of Cox-1. We found Cox-2 expression after exposure to P or Q10 led to a significant increase in expression of Cox-2 gene compared with untreated cells. Similarly, exposure to CP alone also considerably increased Cox-2 gene expression. On the contrary, coexposure of the cells to CP and Q10 led to significant decrease of Cox-2 expression compared to control cells (∗p<0.05). Furthermore, when CP was combined with P and/or Q10, Cox-2 gene expression was maintained at the levels of the control unexposed cells (Figure 6).Figure 6
mRNA levels of inflammatory related genes in HuH7 cells after treatments for 48 h. Each value represents themean±SE of three experiments. n=3 (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) vs. control.The expression ofTNF-α was assessed in the HuH7-treated cells after 48 h. Current results indicated that expression of TNF-α was significantly upregulated after treatment with P, Q10, P+ Q10, CP + P, and CP+ Q10 compared to untreated cells (∗p<0.05, ∗∗p<0.01). Furthermore, when CP was combined with P and Q10, there was an insignificant downregulation of TNF-α expression compared to CP alone. The expression of IFN-γ was considerably increased after exposure to CP. However, a decrease in IFN-γ gene expression was observed after exposure to P alone or with Q10 and CP with P or with Q10. CP combination with P and Q10 resulted in similar level s of IFN-γ expression as in the control untreated cells. Assessment of IL-1α expression showed that exposure to P alone or with CP and/or P+Q10 caused significant downregulation of IL-1α expression compared to untreated control cells (∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001). In addition, P alone or with Q10 led to significant reduction in IL-1α expression compared to CP (#p<0.05) (Figure 6). This suggests that coexposure of the cells to P alone or with Q10 suppresses both basal and CP-mediated IL-1α expression. Figure 6 shows the results obtained for IL-1β expression. Upon exposure of the HuH-7 cells to P, P+Q10, and CP+ P+Q10, expression of IL-1β was significantly downregulated when compared with the untreated cells (∗p<0.05, ∗∗p<0.01). Furthermore, considerable but not statistically significant downregulation of the gene expression was observed after cotreatments with CP+P, CP+Q10, and CP+P+Q10 compared to CP alone (Figure 6). This suggests that coexposure of the cells to P alone or with Q10 suppresses both basal and CP-mediated IL-1β gene expression.Analysis ofIL-6β expression after treatments of HuH-7 with CP, P, Q10, and P+ Q10 for 48 h indicated a significantly increased IL-6β gene expression compared to untreated cells (∗∗p<0.01, ∗∗∗p<0.001). In contrast, treatments of the cells with CP+P, CP+Q10, and CP+P+ Q10 led to significantly decreased compared to CP (#p<0.05, ##p<0.01) (Figure 6). This finding indicates coexposure of the cells to P and/or Q10 suppresses both basal and CP-mediated IL-6β gene expression. The obtained results from IL-12β analysis showed that expression of IL-12β was significantly downregulated by exposure of the cells to P, Q10, P+Q10, CP+P, and CP+P+Q10 compared to control cells (∗∗p<0.01, ∗∗∗p<0.001) (Figure 6). The expression of IL-22 gene was unaffected by all the compounds alone or in combination except for CP+Q10 exposure.


## 3.1. P and Q10 Exhibit Lower Cytotoxicity vs. CP

MTT assay was performed to determine the cytotoxicity of CP and impact of P and Q10 on CP’s cytotoxicity, postexposure to the compounds. We observed dose-dependent decrease of cell survival for all the compounds. Both P and Q10 had similar impact on cell viability at 5μg/ml compared to unexposed cells while CP significantly induced cell viability at 5 μg/ml compared to the control unexposed cells. Similarly, we found that coexposure of the HuH-7 resulted in less cytotoxic effect compared to CP (Table 1).Table 1
Cell viability of HuH-7 cells after treatment with CP, PIP, CoQ-10, and PIP+CoQ-10 for 48 hrs as evaluated by MTT assay. Each value represents themean±SE (n=3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) compared with control.
TreatmentsConcentrations (μg/ml)AverageViability (%)IC50 (μg/ml)Control—0.557±0.001100CP50.373±0.00367.05∗∗10100.273±0.00349.1∗∗∗150.175±0.00431.5∗∗200.138±0.00624.8∗∗∗250.093±0.00216.6∗∗∗PIP50.523±0.1293.916100.382±0.0768.6150.275±0.00749.4∗∗200.231±0.0241.5∗∗250.186±0.0233.4∗Q1050.44±0.00178.915100.368±0.0166∗150.277±0.0249.7∗200.206±0.0137∗∗250.161±0.0228.9∗PIP+Q1050.42±0.0775.418100.387±0.0469.4150.338±0.0560.7200.258±0.0546.4250.231±0.0541.4


## 3.2. P and Q10 Enhance Permeabilisation of HuH-7 Cell Membrane

Findings from LDH assay indicated a significant loss of membrane integrity after exposure of the cells to all investigated compounds when compared to untreated cells after 48 h (Figure1). However, combination of CP+P or CP+Q10 or CP with both P and Q10 significantly increased permeability of the cell membrane compared to CP. This indicates P/Q10 seems to enhance cell membrane permeabilisation effect of CP alone.Figure 1
Effect of CP, PIP, and CoQ-10 on LDH % in HuH-7 cell lines when treated for 48 h (∗p<0.05, ∗∗∗p<0.001) compared to the control group (#p<0.05, ##p<0.01) compared with CP.


## 3.3. Induction of (ROS)

A significant increase in ROS level posttreatment with CP compared with control unexposed cells (p<0.05). Contrastingly, single exposure to either of P or Q10 alone or combination of both compounds resulted in significant suppression of ROS generation compared to control cells (p<0.05). Furthermore, coexposure of the cells to either of CP+P, CP+Q10, and CP+P+ Q10 significantly suppressed ROS generation compared to exposure to CP alone (Figures 2(a) and 2(b)).Figure 2
(a) The production of ROS in HuH-7 cells as stained with fluorescence dye (DCFH-DA) after treatments. Bar: 400μm. (b) Induction of ROS levels in HuH-7 after treatment with CP, PIP, and CoQ-10 for 48 h. Each value represents the mean±SE (n=3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) compared with control (#p<0.05, ##p<0.01, ###p<0.001) compared with CP.
(a)(b)


## 3.4. Catalase (CAT) Activity

Based on the suppressed ROS generation observed above, we proceeded to assess catalase activity in the cell postexposure to the compounds. Our findings showed significant reduction in CAT activity posttreatment with P, CP+Q10, and CP+P+Q10 was significantly reduced after 48 h (∗p<0.05, ∗∗p<0.01) (Figure 3).Figure 3
CAT levels in HuH-7 after treatment with CP, PIP, Q-10, and in combination of CP with each compound for 48 hr. Data present represents themean±SE (n=3) (∗p<0.05, ∗∗p<0.01) compared with untreated control.


## 3.5. Superoxide Dismutase (SOD)

This enzyme catalyzes the conversion of superoxide radical into less-toxic hydrogen peroxides to help healthy cells actively fight oxidative stress. After treatment, a significant drop in activity of SOD due to CP exposure was observed compared to the control cells (∗∗p<0.01). In addition, reduction in SOD activity was observed after treatment with other investigated compounds compared to control although these were not significant. Similarly, a nonsignificant increase in SOD activity was observed after treatment with CP+P, CP+Q10, and CP with both P and Q10 compared to CP alone (Figure 4).Figure 4
SOD levels in HuH-7 after treatment with CP, PIP, CoQ-10, and in combination of CP with each compound for 48 hr. Data present represents themean±SE (n=3) (∗p<0.05) compared with untreated control.


## 3.6. Glutathione (GSH)

Glutathione (GSH) plays a crucial function in removal of many reactive species. HuH-7 cells were exposed to CP, P, Q10, and in combination with CP. Our result as shown in (Figure5) indicated nonsignificant reduction in GSH level when exposed to CP, P, and Q10 but there was significant decrease in GSH level after treatment with combination of P+Q10 and combination of CP+P and CP+P+ Q10 compared to CP.Figure 5
GSH levels in HuH-7 after treatment with CP, PIP, Q-10, and in combination of CP with each compound for 48 hr. Data present represents themean±SE (n=3) (∗∗p<0.01) compared with untreated control (#p<0.05, ##p<0.01) compared with CP.


## 3.7. Gene Expression

### 3.7.1. Inflammatory Marker Genes at Transcriptional Level

Gene expression ofCox-1 was found higher in cells exposed to CP alone. However, coexposure of the cells to CP and Q10 resulted in significant reduction in Cox-1 gene expression (#p<0.05). We found that coexposure of the cells to CP and either of P or P+Q10 as well as single exposure to P alone resulted in considerable reduction of Cox-1 expression (Figure 6). Assessment of Cox-2 expression produced a contrasting finding to that of Cox-1. We found Cox-2 expression after exposure to P or Q10 led to a significant increase in expression of Cox-2 gene compared with untreated cells. Similarly, exposure to CP alone also considerably increased Cox-2 gene expression. On the contrary, coexposure of the cells to CP and Q10 led to significant decrease of Cox-2 expression compared to control cells (∗p<0.05). Furthermore, when CP was combined with P and/or Q10, Cox-2 gene expression was maintained at the levels of the control unexposed cells (Figure 6).Figure 6
mRNA levels of inflammatory related genes in HuH7 cells after treatments for 48 h. Each value represents themean±SE of three experiments. n=3 (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) vs. control.The expression ofTNF-α was assessed in the HuH7-treated cells after 48 h. Current results indicated that expression of TNF-α was significantly upregulated after treatment with P, Q10, P+ Q10, CP + P, and CP+ Q10 compared to untreated cells (∗p<0.05, ∗∗p<0.01). Furthermore, when CP was combined with P and Q10, there was an insignificant downregulation of TNF-α expression compared to CP alone. The expression of IFN-γ was considerably increased after exposure to CP. However, a decrease in IFN-γ gene expression was observed after exposure to P alone or with Q10 and CP with P or with Q10. CP combination with P and Q10 resulted in similar level s of IFN-γ expression as in the control untreated cells. Assessment of IL-1α expression showed that exposure to P alone or with CP and/or P+Q10 caused significant downregulation of IL-1α expression compared to untreated control cells (∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001). In addition, P alone or with Q10 led to significant reduction in IL-1α expression compared to CP (#p<0.05) (Figure 6). This suggests that coexposure of the cells to P alone or with Q10 suppresses both basal and CP-mediated IL-1α expression. Figure 6 shows the results obtained for IL-1β expression. Upon exposure of the HuH-7 cells to P, P+Q10, and CP+ P+Q10, expression of IL-1β was significantly downregulated when compared with the untreated cells (∗p<0.05, ∗∗p<0.01). Furthermore, considerable but not statistically significant downregulation of the gene expression was observed after cotreatments with CP+P, CP+Q10, and CP+P+Q10 compared to CP alone (Figure 6). This suggests that coexposure of the cells to P alone or with Q10 suppresses both basal and CP-mediated IL-1β gene expression.Analysis ofIL-6β expression after treatments of HuH-7 with CP, P, Q10, and P+ Q10 for 48 h indicated a significantly increased IL-6β gene expression compared to untreated cells (∗∗p<0.01, ∗∗∗p<0.001). In contrast, treatments of the cells with CP+P, CP+Q10, and CP+P+ Q10 led to significantly decreased compared to CP (#p<0.05, ##p<0.01) (Figure 6). This finding indicates coexposure of the cells to P and/or Q10 suppresses both basal and CP-mediated IL-6β gene expression. The obtained results from IL-12β analysis showed that expression of IL-12β was significantly downregulated by exposure of the cells to P, Q10, P+Q10, CP+P, and CP+P+Q10 compared to control cells (∗∗p<0.01, ∗∗∗p<0.001) (Figure 6). The expression of IL-22 gene was unaffected by all the compounds alone or in combination except for CP+Q10 exposure.


## 3.7.1. Inflammatory Marker Genes at Transcriptional Level

Gene expression ofCox-1 was found higher in cells exposed to CP alone. However, coexposure of the cells to CP and Q10 resulted in significant reduction in Cox-1 gene expression (#p<0.05). We found that coexposure of the cells to CP and either of P or P+Q10 as well as single exposure to P alone resulted in considerable reduction of Cox-1 expression (Figure 6). Assessment of Cox-2 expression produced a contrasting finding to that of Cox-1. We found Cox-2 expression after exposure to P or Q10 led to a significant increase in expression of Cox-2 gene compared with untreated cells. Similarly, exposure to CP alone also considerably increased Cox-2 gene expression. On the contrary, coexposure of the cells to CP and Q10 led to significant decrease of Cox-2 expression compared to control cells (∗p<0.05). Furthermore, when CP was combined with P and/or Q10, Cox-2 gene expression was maintained at the levels of the control unexposed cells (Figure 6).Figure 6
mRNA levels of inflammatory related genes in HuH7 cells after treatments for 48 h. Each value represents themean±SE of three experiments. n=3 (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001) vs. control.The expression ofTNF-α was assessed in the HuH7-treated cells after 48 h. Current results indicated that expression of TNF-α was significantly upregulated after treatment with P, Q10, P+ Q10, CP + P, and CP+ Q10 compared to untreated cells (∗p<0.05, ∗∗p<0.01). Furthermore, when CP was combined with P and Q10, there was an insignificant downregulation of TNF-α expression compared to CP alone. The expression of IFN-γ was considerably increased after exposure to CP. However, a decrease in IFN-γ gene expression was observed after exposure to P alone or with Q10 and CP with P or with Q10. CP combination with P and Q10 resulted in similar level s of IFN-γ expression as in the control untreated cells. Assessment of IL-1α expression showed that exposure to P alone or with CP and/or P+Q10 caused significant downregulation of IL-1α expression compared to untreated control cells (∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001). In addition, P alone or with Q10 led to significant reduction in IL-1α expression compared to CP (#p<0.05) (Figure 6). This suggests that coexposure of the cells to P alone or with Q10 suppresses both basal and CP-mediated IL-1α expression. Figure 6 shows the results obtained for IL-1β expression. Upon exposure of the HuH-7 cells to P, P+Q10, and CP+ P+Q10, expression of IL-1β was significantly downregulated when compared with the untreated cells (∗p<0.05, ∗∗p<0.01). Furthermore, considerable but not statistically significant downregulation of the gene expression was observed after cotreatments with CP+P, CP+Q10, and CP+P+Q10 compared to CP alone (Figure 6). This suggests that coexposure of the cells to P alone or with Q10 suppresses both basal and CP-mediated IL-1β gene expression.Analysis ofIL-6β expression after treatments of HuH-7 with CP, P, Q10, and P+ Q10 for 48 h indicated a significantly increased IL-6β gene expression compared to untreated cells (∗∗p<0.01, ∗∗∗p<0.001). In contrast, treatments of the cells with CP+P, CP+Q10, and CP+P+ Q10 led to significantly decreased compared to CP (#p<0.05, ##p<0.01) (Figure 6). This finding indicates coexposure of the cells to P and/or Q10 suppresses both basal and CP-mediated IL-6β gene expression. The obtained results from IL-12β analysis showed that expression of IL-12β was significantly downregulated by exposure of the cells to P, Q10, P+Q10, CP+P, and CP+P+Q10 compared to control cells (∗∗p<0.01, ∗∗∗p<0.001) (Figure 6). The expression of IL-22 gene was unaffected by all the compounds alone or in combination except for CP+Q10 exposure.


## 4. Discussion

Some side effects hampering the application or administration of CP in cancer treatment includes associated oxidative stress and inflammation [19]. In this study, we evaluated the impact of two naturally occurring compounds, P and Q10, as protective agents that can mediate suppression of inflammation and oxidative stress induced by CP. One of the uses of CP is in treatment of liver cancer. The first step in the activation reaction of CP occurs in the liver microsomal oxidation system in a process that produces 4-hydroxy-CP, a cytotoxic metabolite that diffuses from liver cells into the plasma and then to other organs in the body. As such, we have selected the HuH-7 cell line, as a hepatocellular carcinoma model [20]. Findings from this study showed that HuH-7 cell treatment with CP significantly reduced cell viability with an IC50 of 10 μg/ml higher than the IC50 of P and Q10 alone or in combination, indicating CP was more cytotoxic compared to P and Q10. This reiterates the cytotoxicity of CP as supported by previous studies in hepatocarcinoma cells [21–23]. One of the toxicity mechanism of CP is through generation of intracellular ROS, which causes oxidative stress. Investigation of ROS generation upon CP treatment showed that the HuH-7 cells underwent significant oxidative stress based on the increased ROS generation in comparison to the control untreated cells. The increased ROS generation by CP led to our assessment of some oxidative markers such as catalase, SOD, and GSH. We found that CP treatment caused reduction in SOD activities while catalase activity and GSH levels were unaffected. This finding is supported by Germoush and Mahmoud [24], who showed CP induced suppression of SOD activity and GSH levels in a rat model. Oxidative stress within a cell is a measure of the balance between pro- and antioxidant activities. Increased ROS in a normal cell is followed by increased levels and activities of antioxidants such as catalase, SOD, and GSH to metabolize the ROS. SOD will convert ROS to H2O2 while catalase converts the H2O2 into oxygen and water [25]. However, inadequate levels or activities of these antioxidants that can cope with the generated ROS as observed in CP-treated cells result in detrimental oxidative stress [26, 27]. P and Q10 were both found to significantly suppress ROS generation alone or during coexposure. Similarly, we found P and Q10 suppressed CP-induced ROS generation in HuH-7 cells alone or when they are both coexposed with CP. This finding may be because of both P and Q10 antioxidant activities. P is initially oxidized to form a catechol that has an antioxidant activity that is known to be neuroprotective [28]. Q10 is an important electron acceptor during ATP synthesis, making it an important compound for normal cellular energy homeostasis. In addition, Q10 is effective against oxidation that results in modifications of organic and genetic materials. Q10 level often declines in some diseases correlated with increased ROS generation and activity and deficiency of Q10 results in decreased cell efficiency consequent upon respiratory chain dysfunction, which is characterized with insufficient production of high-energy compounds [29]. The finding in our study indicates that P and/or Q10 did not influence SOD activity while they suppressed catalase activity and GSH level. This finding shows that both P and Q10 maintained the antioxidant and prooxidant balance since ROS generation is suppressed requiring no activity of the antioxidant parameters.Intracellular ROS can induce activation of cellular pathways mediating activities of redox sensitive transcription factors like nuclear factor-κβ (NF-κβ) which then activate expression of proinflammatory proteins like cox-2, IL-1β, IL-6, and TNF-α [10, 30]. Induction of proinflammatory cytokines like Cox-2 can lead to increased levels of prostaglandin, resulting in recruitment of more inflammatory cells followed by and stimulation of inflammatory responses and generation of ROS [31]. Similarly, this sustained loop of inflammation is seen during induction of IL-6β resulting from NFκβ activation. IL-6β binds its receptor, IL-6R, which activates STAT-3, a transcription factor regulating transcription of NFκβ with further expression of IL-6β [32].We found that CP treatment alone did not induce gene expression of any of the proinflammatory cytokines we evaluated except forIL-6β. However, coexposure of the HuH-7 cells to Q10 and CP caused significant suppression of basal Cox-2 and TNF-α gene expression while coexposure of the cells to CP and P with Co-Q10 suppressed basal IL-1β gene expression. Q10 also suppressed CP-induced expression of Cox-1. Similarly, P and CP suppressed basal expression of IL-6β and IL-12β, while P and Q10 suppressed CP-induced expression of IL1-α. These findings suggest that anti-inflammatory effect Q10 and P of CP-mediated inflammation is due to inhibition of NFκβ pathway. NFκβ activation controls the regulation of myriads of inflammatory proteins including the aforementioned proteins that were inhibited in this study [33]. These findings are in line with other studies that have investigated anti-inflammatory activities of natural compounds on CP. Mansour et al. [34] showed that genistein inhibited CP-induced Cox-2 expression. Similarly, Vernonia cinerea was also reported to suppress CP-induced TNF-α gene expression in a mouse model [19]. Contrastingly, the anti-inflammatory roles of P and Q10 as illustrated in this study are supported by those of other studies. P has been shown to suppress protein expression of inflammatory proteins like Cox-2, IL-6, and IL-8 through suppression of ROS in HaCaT cells [31]. P also downregulates gene expression of IL-1β in human osteoarthritis chondrocytes [35]. Also, IL-1β-mediated expression of Cox-2 gene and protein was abrogated. In a similar manner, there are studies showing anti-inflammatory activities of Q10, supporting our findings here on the impact of Q10 on CP-mediated inflammation. Q10 has been demonstrated to mediate its antiapoptotic and anti-inflammatory activities, via redox-dependent mechanisms. For instance, it has been shown that Q10 supplementation causes reduction in plasma levels of IL-6, TNF-α, and C-reactive protein (CRP) [36].


## 5. Conclusion

Our findings shows that Q10 and/or P induced suppression of ROS generation mediated by CP. We also found that Q10 and P suppressed CP-induced inflammation by inhibiting gene expression-specific inflammatory cytokine. This anti-inflammatory role of Q10 and P is likely linked to their antioxidant mechanism. Findings from this study thus show that Q10 and P may have protective effect on oxidant and inflammatory side effects of CP.



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*Source: 8495159-2022-07-13.xml*