Therapeutic potential of kaempferide against paraquat instigated cardiac toxicity in rats

Muhammad Umar Ijaz; Syeda Sania Zahara; Moazama Batool; Mikhlid H. Almutairi; Ayesha Ishtiaq; Asma Ashraf

doi:10.1016/j.jksus.2023.102980

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01 2023

:36;

102980

doi:

10.1016/j.jksus.2023.102980

Therapeutic potential of kaempferide against paraquat instigated cardiac toxicity in rats

Muhammad Umar Ijaz^{^a}, Syeda Sania Zahara^{^a}, Moazama Batool^{^b}, Mikhlid H. Almutairi^{^c}, Ayesha Ishtiaq^{^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 College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China

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

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

Received: 2023-7-1, Accepted: 2023-10-28,

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

Paraquat (PQ) is an effective herbicide however, due to its adverse effects on different organs including heart, it is considered as highly toxic to human beings. Kaempferide (KMF) is a plant-based flavonoid with conspicuous pharmacological properties. This experiment was executed to evaluate the palliative actions of KMF on PQ prompted cardiac toxicity in rats. Twenty-four rats were apportioned into 4 equal groups which were designated as control, toxicant (PQ treated), Co-treated (PQ + KMF) and KMF group. After 30 days of treatment, PQ intoxication resulted in a remarkable reduction in antioxidant enzymes activities which include, glutathione reductase (GSH), glutathione S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione disulfide reductase (GSR), whereas an elevation was observed in reactive oxygen species (ROS), & malondialdehyde (MDA) as well as hydrogen peroxide (H₂O₂) level. Furthermore, concentrations of cardiac injury markers, creatinine phosphokinase (CPK), creatine kinase-myoglobin binding (CK-MB), & lactate dehydrogenase (LDH), as well as troponin I were increased in response to PQ treatment. Moreover, inflammatory cytokines such as tumour necrosis factor alpha (TNF-α), nuclear factor-kappa B (NF-κB), and interleukin-6 (IL-6), interleukin-1 beta (lL-1β), and cyclooxygenase-2 (COX-2) levels were augmented in PQ intoxicated group. PQ exposure reduced the gene expression of cardiac anti-apoptotic markers (Bcl-2), but the gene expression of apoptotic marker (caspase-9, caspase-3 and Bax) was increased. Histopathological damages were also observed in toxicant (PQ) exposed group. However, the administration of KMF significantly palliated PQ induced aforementioned disruptions. In the light of these findings, it is concluded that KMF is a promising bioactive compound that may be used as a curative agent against PQ instigated cardiac damage due to its marvelous antioxidant, anti-apoptotic, anti-inflammatory as well as cardioprotective potential.

Keywords

Kaempferide

Paraquat toxicity

Cardiac injury

Oxidative stress

Inflammation

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1 Introduction

Paraquat is a nitrogen-based herbicide which is used to control the progression of different herbs by interrupting electron transport chain during the process of photosynthesis (Qian et al., 2009). According to Dinis-Oliveira et al. (2008), In poorly developed countries such as Asia, the intoxication of PQ considered as a major health threat to living organisms. The rate of fatalities due to intoxication of paraquat depends upon the duration of exposure and route (ingestion, intravenous, intraperitoneally) of exposure, for instance, PQ exposure through skin and mouth for 3.5 h can cause immediate death (Nikdad et al., 2020). Animals as well as humans are highly susceptible to both acute and chronic PQ intoxication.

Primary signs of PQ intoxication include respiratory failure, cardiac damages, hepato-renal injuries, adrenal gland malfunctioning, spleen inflammation as well as dysfunctioning of immune system (Dinis-Oliveira et al., 2008). It is documented that reactive oxygen species (ROS) are major culprit behind organ toxicities instigated by PQ. ROS are byproducts of cellular metabolism and responsible for proper modulation of cell signaling, cell survival, inflammatory responses as well as cell differentiation. However, excessive generation of free radical without proper control can result in serious injuries (Zeinvand-Lorestani et al., 2015). It is reported that paraquat produces large amounts of ROS which ultimately elevates level of oxidative stress and causes cellular damages (McCormack et al., 2005).

PQ consumption results in rapid systemic absorption which damages multiple organs (Onyon and Volans, 1987). Cardiovascular damage is one of the overt complications of PQ poisoning. Abovementioned complications lead toward various cardiogenic events such as arrhythmias, heart failure, and cardiac arrest (Fukushima et al., 2010). Numerous investigations documented that PQ intoxication surge up the level of inflammatory markers as well as ROS generation (Lin et al., 2021). Another study delineated that PQ prompted cardiac damages by intensifying vascular permeability which reduces the fractional shortening and cardiac remodeling in animals (Ahmad et al., 2021).

Flavonoids are known for their distinct ability to function as antioxidant as well as wide range of other pharmacological activities. KMF (methoxy flavone) is an emerging plant-based flavonoid which is extracted from the roots of Alpina officinarum, commonly known as lesser galangal (Matsuda et al., 2009). KMF exhibits excellent antioxidant properties and has been found to manifest other as well as pharmacological activities such as anti-inflammatory, anti-cancerous & antihypertensive (Maruyama et al., 2009). The current research was executed to assess the cardioprotective abilities of KMF against PQ instigated cardiac injury.

2 Materials and methods

2.1

2.1 Chemicals

Both PQ and KMF were procured from Sigma-Aldrich (Merch, Germany).

2.2

2.2 Animals

Twenty-four albino rats aged from 10 to 12 weeks having weight between 180 and 200 g were placed at the research station of University of Agriculture, Faisalabad. A light/dark cycle of 12 h was strictly maintained. Prior to the trials animals were underwent to acclimatization to the laboratory conditions for one week and provided them food pellets as well as tap water while temperature was adjusted at 20–26 °C. The animals were handled as per the guidelines of European Union of animal care and experimentation approved (CEE Council 86/ 609).

2.3

2.3 Experimental layout

Animals were apportioned into equal sized groups having six rats in each. All the groups were assigned treatment as follow: Group 1st was termed as untreated group. Group 2nd was provided with (5 mg/kg) of PQ. Group 3rd was given with combine treatment of PQ (5 mg/ kg) + KMF (20 mg/ kg) while the group 4th was given with KMF (20 mg/ kg) only for 30 days with the help of an oral gavage. On the 30th day of trial, rats were made unconscious with the help of ketamine (60 mg/kg) + xylazine (6 mg/kg) then decapitated and blood was collected in blood sample vial for further analysis. Single piece of heart was fixed in a buffer solution of 10 % formalin for histopathological evaluations while the other piece was kept in a zip-lock bag and freeze at −20 °C to access various biochemical parameters.

2.4

2.4 Evaluations of antioxidants enzymes

The protocol described by Chance and Maehly (1955) was utilized to assess the CAT activity. Biochemical activity of SOD was measured by using strategies described by Kakkar et al., (1984). Sedlak and Lindsay (1968) technique was employed to determine GSH activity. The GSR activity was ascertained by using the protocol elaborated by Carlberg and Mannervik (1975). The methodology of Couri and Abdel-Rahman (1979) was used for the analysis of GST activity. The protocol elaborated by Lawrence and Burk (1976) was used to evaluate GPx activity.

2.5

2.5 Evaluation of oxidative stress

Ohkawa et al. (1979) method was employed to determined MDA level. A technique described by Hayashi et al. (2007) was utilized to assess the level of ROS·H₂O₂ concentration was evaluated using Pick and Keisari's technique (1981).

2.6

2.6 Evaluation of cardiac injury markers

Bais and Philcox (1994) strategy was employed to assess the activity of lactate dehydrogenase (LDH) was measured in serum. With the help of fully automated analyzer Cobas-Integra 400, the activity of CPK and CK-MB was determined through enzymatic assay as well as immune inhibition assay respectively (Tietz et al., 1983). The protocol elaborated by Panteghini et al. (2004) was employed to evaluate the activity of troponin I.

2.7

2.7 Inflammatory markers analysis

The inflammatory markers level such as IL-6, NF-ƙB, TNF-α and IL-1β as well as COX-2 activity were assessed using rat ELISA kits (Shanghai-Biotech. Ltd).

2.8

2.8 Assessment of apoptotic proteins

The standard ELISA kits were used to assess the level of caspase-9, Bcl-2, caspase-3, and Bax according to the manufacturer’s instruction.

2.9

2.9 Histopathological observation

The cardiac tissues were fixed in 10 % formaldehyde following the process of dehydration with different alcoholic reagents. A rotatory microtome was used to cut the dehydrated pieces into 4–5 μm. The slices were stained using hematoxylin-eosin stain and then observed under light microscope while the photographs were captured with the help of MoticTM megapixel camera.

2.10

2.10 Statistical analysis

The findings of the current investigation were displayed in tables as Means ± SEM by using ONE way ANOVA. Minitab (Version 17) software was used to compare the treatments by applying Tukey’s test p < 0.05 was adjusted as the level of significance.

3 Results

3.1

3.1 Effect of PQ and KMF on antioxidant enzymes

PQ exposure considerably (p < 0.05) suppressed the activities of GST, GSR, CAT, GPx, SOD, & level of GSH when juxtaposed with untreated group. However, KMF treatment exhibited prominent (p < 0.05) escalation in enzymatic antioxidant capacity and GSH level in PQ + KMF treated group in contrast with PQ-intoxicated group, which shows the antioxidant ability of KMF. However, no remarkable change was noticed in the antioxidant enzymes activity in KMF (only) as well as untreated group (Table 1).

Table 1 Effects of PQ and KMF on antioxidant enzymes (CAT, GPx, SOD, GSR, GST, GSH).

Groups	CAT (U/mg protein)	GPx (μmole)	SOD (nmoL)	GSR (Nm NADPH oxidized/min/mg tissue	GST (mg/dL)	GSH (nM /min/mg protein)
Control	10.62 ± 0.43^a	25.71 ± 0.82^a	7.83 ± 0.06^a	7.05 ± 0.11^a	31.71 ± 0.89^a	17.47 ± 0.19^a
PQ	4.84 ± 0.07^c	7.81 ± 0.16^c	2.93 ± 0.11^c	2.64 ± 0.05^c	10.55 ± 0.43^c	4.36 ± 0.38^b
PQ + KMF	7.52 ± 0.09^b	15.98 ± 1.02^b	6.45 ± 0.16^b	6.26 ± 0.09^b	25.52 ± 0.64^b	12.33 ± 0.65^c
KMF	10.67 ± 0.42^a	25.82 ± 0.87^a	7.85 ± 0.07^a	7.08 ± 0.12^a	32.59 ± 0.79^a	17.68 ± 0.32^a