Curative effects of kaempferide on cadmium-instigated hepatotoxicity in male albino rats

Muhammad Umar Ijaz; Sidra Aziz; Ali Hamza; Moazama Batool; Saad Alkahtani; Mian Nadeem Riaz; Asma Ashraf

doi:10.1016/j.jksus.2023.102885

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Original article

11 2023

:35;

102885

doi:

10.1016/j.jksus.2023.102885

Curative effects of kaempferide on cadmium-instigated hepatotoxicity in male albino rats

Muhammad Umar Ijaz^{^a}, Sidra Aziz^{^a}, Ali Hamza^{^a}, Moazama Batool^{^b}, Saad Alkahtani^{^c}, Mian Nadeem Riaz^{^d}, Asma Ashraf^{^e,⁎}

a Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad 38040, Pakistan

b Department of Zoology, Govt. College Women University, Sialkot 51310, Pakistan

c Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

d Texas A & M University, College Station, TX 2476, USA

e Department of Zoology, Government College University, Faisalabad 38000, Pakistan

⁎Corresponding author. asmaashraf@gcuf.edu.pk (Asma Ashraf)

Received: 2023-5-25, Accepted: 2023-8-31,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Cadmium (Cd) is an environmental and industrial toxicant that possess the ability to cause severe health issues to humans and animals. The flavonol, kaempferide (KF), exhibits multiple pharmacological potentials. Therefore, this investigation was designed to estimate the mitigative effect of KF against Cd instigated hepatic damage in rats. 24 albino rats (male) were kept in 4 different groups. The group-1 was designed as untreated/control group, group-2 was orally provided with Cd (5 mg/kg), group-3 was orally co-administrated with Cd (5 mg/kg) along with KF (20 mg/kg) and group-4 was supplemented with KF (20 mg/kg) during 30 days of trial. Cd administration significantly escalated ALT, ALP as well as AST levels. A substantial decline in SOD, GST, POD, CAT, GSR activities and GSH content was observed due to Cd exposure along with an elevation in TBARS and H₂O₂ levels. Moreover, Cd administration elevated NF-kB, IL-6, TNF-α as well as IL-1β levels in addition to the activity of COX-2. Furthermore, the levels of Bax, Caspase-9 as well as Caspase-3 were escalated, besides the level of Bcl-2 was decreased after Cd exposure. A significant increase in histopathological damages after Cd administration is a clear indication of liver damage. However, co-treatment of KF with Cd potential recovered all the damages and significantly reduced the biochemical and histological variations in rat liver. Overall results indicated that KF has efficient anti-oxidant properties, which mitigated Cd instigated hepatic damage in rats.

Keywords

Cadmium

Flavonoid

Hepatotoxicity

Kaempferide

Antioxidant

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1 Introduction

Increasing human activities significantly disturbed the universal cycle of metalloids and heavy metals i.e., cadmium (Cd) like toxic elements. Cd is among the major environmental and industrial toxicants (Kadry and Megeed, 2018). Its prevalent existence in natural environment and its application in daily life activities (Ma et al., 2019) cause inevitable exposure to humans (Alshatwi et al., 2014). It is profoundly present in products of polyvinyl chloride, tires, photo-cells, heating elements, electrical conductors and automobile radiators (Mead, 2010). According to El-Maraghy and Nassar (2011) cigarette smoking is one of the main source of Cd exposure. Furthermore, the use of Cd containing phosphate fertilizers may elevate Cd level in crops and natural environment (Newbigging et al., 2015).

Cd can potentially accumulate in different body organs particularly in liver (Rahimzadeh et al., 2017). Cd induces functional damages in several organs like lung, pancreas, testes, kidneys, heart and liver at cellular level (Cuypers et al., 2010). Exposure to Cd may cause different hepatic variations (Othman et al., 2014). At lower doses, Cd induces several pathological alterations in hepatic tissues. Several investigations revealed that Cd can change anti-oxidant status, which leads to oxidative damage (El-Habit and Abdel Moneim, 2014). The mechanism through which Cd damages the tissues is the reactive oxygen species (ROS) generation, which react with cellular biomolecules and causes DNA damage, lipid peroxidation, membrane protein injury, apoptosis and reduced anti-oxidant status (Stohs et al., 2000).

Growing evidences suggest that the use of phytochemicals to treat various ailments is getting traction (Flora et al., 2007). An O-methylated flavonol, kaempferide (KF) is a naturally occurring flavone, which is reported in Kaempferia galangal (Jiao et al., 2017). It shows anti-carcinogenic, anti-inflammatory, anti-oxidant (Zhang et al., 2013), anti-bacterial (Eumkeb et al., 2012) and neuroprotective potentials (Wang et al., 2016). Nevertheless, there is no literature available discussing the therapeutic role of KF against liver damage. By keeping these attributes in mind, this research was designed to estimate the mitigative role of KF on Cd instigated hepatic damage in rats.

2 Materials and methods

2.1

2.1 Chemicals

Cd was bought from Sigma Aldrich (Germany) and KF was bought from Merck (Germany).

2.2

2.2 Animals

Twenty-four male albino rats having weight 180 ± 20 g (6–8 weeks old) were housed in the animal house at the University of Agriculture, Faisalabad (UAF). The animals were placed under controlled conditions at 25 ± 2 °C for 12 hrs. light/dark periods. Throughout the experimental duration, the rats were given tap water and standard diet. Rats were acclimatized for 7 days before the start of the experiment. Rats were treated and handled in compliance with the instructions of the European union of animal care and experimentation (CEE Council 86/ 609).

2.3

2.3 Experimental layout

The animals were allocated into 4 equal groups and housed in separate cages. Animals of group-1 (control) were provided with tap water. Group-2 was intoxicated with Cd (5 mg/kg). Group-3 was treated with Cd (5 mgkg⁻¹) and KF (20 mgkg⁻¹) orally for about 30 days. Group-4 orally received the dose of KF (20 mgkg⁻¹). After 30 days, the animals were made unconscious and beheaded. Blood was gathered in heparinized tubes for further assessment. The liver was excised and divided into 2 equal parts, one half was stored in zipper bags for biochemical observation at −80 °C. The homogenization of hepatic tissues was performed with the chilled phosphate buffer saline (25 mM; pH: 7.4) at 11,000 g for 20 min by using homogenizer. Laterally, the homogenate was used to estimate anti-oxidative enzymes & inflammatory markers. The second half was preserved in 10 percent formalin for histological evaluation.

2.4

2.4 Biochemical evaluation

CAT activity was evaluated calorimetrically by means of dichromate-acetate acid reagent (Sinha (1972). The activity of POD was measured through a standard technique exemplified by Chance and Maehly (1955). The action of SOD was determined using the technique of kakkar et al. (1984). GST activity was computed by the technique of Habig et al., 1974. The evaluation of GSH content was appraised via the protocol of Jollow et al. (1974). GSR content was determined through the procedure of Carlberg and Mannervik (1975). The technique of Jiang et al. (1992) was used to evaluate the TBARS level·H₂O₂ level was appraised via the procedure of Pick and Keisari (1981).

2.5

2.5 Hepato-serum markers analysis

The levels of ALP, AST and ALT were analyzed by using the commercially available kits, bought from Wiesbaden (Germany).

2.6

2.6 Inflammatory biomarkers analysis

Inflammatory markers (IL-1β, TNF-α, IL-6, NF-κB levels and COX-2 activity) were estimated with ELISA kits (Cusabio Technology Llc, USA) in accordance with the company’s instructions.

2.7

2.7 Apoptotic markers assessment

The levels of apoptotic markers i.e., Caspase-9, Bax, Bcl-2 along with Caspase-3 were estimated by ELISA kits (Cusabio Technology Llc, USA) as directed by the manufacturer.

2.8

2.8 Histopathological observations

Fukuzawa et al. (1996) technique was used for histopathological examination of liver. In first step, samples were rinsed for 24 h in 10% formalin for the fixation of the tissue samples and then passed through different grades of alcohol to dehydrate, cleaned with xylene and embedded in paraffin wax. 5 µm thin sections were sliced by using microtome and then stained with Hematoxylin & Eosin stain. Lastly, the slides were examined using light microscope (Nikon Labophot, Japan) with attached camera (Canon-EOS 200D).

2.9

2.9 Statistical analysis

Data were presented as Mean ± SEM. Values were estimated through the application of one-way ANOVA and comparative measurements between treatments were estimated by applying Tukey’s test. Data estimation was performed using Graph Pad Prism software. Statistics were considered significant at p < 0.05.

3 Results

3.1

3.1 Curative effect of KF on anti-oxidant enzymes

Cd administrated rats demonstrated a notable (p < 0.05) reduction in anti-oxidants i.e., CAT, POD, SOD, GST, GSR activities & GSH content when matched to the control rats. However, the administration of Cd + KF notably improved anti-oxidant enzymes activities & GSH content in contrast to Cd intoxicated rats. Furthermore, in KF only supplemented group the activity of anti-oxidant enzymes was comparable to untreated rats (Table 1).

Table 1 The effect of Kaempferide on CAT, SOD, POD, GST, GSH and GSR activities against cadmium induced hepatotoxicity in rats.

Groups	CAT (U/mg protein)	POD (U/mg protein)	SOD (U/mg protein)	GST (nM/min/mg protein)	GSH (μM/g tissue)	GSR (nM NADPH oxidized/min/mg tissue)
Control	8.98 ± 0.11^a	7.11 ± 0.05^a	5.60 ± 0.12^a	24.71 ± 0.76^a	15.30 ± 0.73^a	3.85 ± 0.08^a
Cd (5 mg/kg)	4.69 ± 0.09^b	3.59 ± 0.05^b	3.11 ± 0.06^b	10.77 ± 0.67^b	8.76 ± 0.38^b	1.49 ± 0.08^b
Cd (5 mg/kg) + KF (20 mg/kg)	8.01 ± 0.09^a	6.80 ± 0.08^a	5.22 ± 0.08^a	20.20 ± 0.58^ac	14.55 ± 0.60^c	3.18 ± 0.09^a
KF (20 mg/kg)	8.71 ± 0.13^a	7.10 ± 0.07^a	5.41 ± 0.18^a	22.85 ± 0.91^c	15.51 ± 0.76^a	3.48 ± 0.15^a