INTRODUCTION:

Stroke is the second leading cause of death and long-term disability in the world¹. In the ischemic stroke, diabetes is one of the major risk factor. The diabetes in ischemic reperfusion state can increase the inflammation, oxidative stress and apotosis induced by reperfusion². There is an increased mortality rate in patients with diabetes associated with cerebrovascular accident (ischemic stroke and intra cerebral hemorrhage) and are at more risk of suffering with organ damage and ischemic events³.

In acute stroke, thrombolysis plays as life saving therapy and helps in reperfusion. Although reperfusion is needed in ischemic stroke, it can exaggerate the condition causing further damage through inflammation and reactive oxygen species released during reperfusion. This can be further augmented by diabetes making the condition more worsen. These implications associated with reperfusion injury have made the active attention to it. The pathological aspects of reperfusion injury are related with oxidative stress, leukocyte infiltration, damage to blood brain barrier, inflammation, nitric oxide release, platelet activation and apoptosis⁴. Worsening of clinical and laboratory outcomes are seen in ischemic reperfusion injury patients with diabetes⁵. The intervention with anti-inflammatory and anti-oxidant agents was thought to be beneficial in treating cerebral ischemia reperfusion injury. Chrysin, a flavonoid extracted mainly from honey and bee propolis was reported to be having many beneficial properties like anti-oxidant, anti-inflammatory, anti diabetic, anti allergic, anti convulsants, anti hypertensive, anti cancer and controlling apoptosis^6,7,8,9. In the present study, we made an attempt to investigate the protective role of Chrysin in cerebral ischemia reperfusion injury in Wistar diabetic rats by employing common carotid arteries occlusion for 30 min and reperfusion for 4 hours.

MATERIAL AND METHODS:

Chemicals:

BCl2, BAX and Hsp90 Rat ELISA Kit (Assyrapro, USA), Rat TNF-α ELISA kit (Assyrapro, USA), Glutathione Assay kit (Sigma Aldrich), Xanthine oxidase Kit(Sigma Aldrich), Rat IL-6 ELISA kit (Eaab, USA), Rat IL-10 ELISA kit (Assyrapro, USA), Rat IL-6 ELISA kit (Eaab, USA), Chrysin and Streptozocine (STZ) procured from Sigma Aldrich, India, Phenazine methosulphate (PMS) (Loba chemicals, India), Thiobarbituric acid (TBA) (Loba chemicals, India), Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) (Sisco chemicals, India), Nitoblue tetrazolium (NBT) (SD fine chemicals, India), O-di anisidine di hydrochloride (Sigma Aldrich, India), 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma Aldrich, India).

Glutamate ELISA Kit (abnova), Aspartage ELISA (abnova). Other chemicals used were of analytical grade supplied from local agencies.

Animals:

Adult Wistar rats (220–310g) were obtained from NIN, Hyderabad, Andhra Pradesh, India. Animals were maintained under a 12/12-hr light/dark cycle, in an ambient temperature (24±1°C) colony room. Animals were provided with a constant supply of food and water. Animal care followed the official governmental guidelines in compliance with the CPCSEA, New Delhi and experimental protocols were conducted at GITAM Institute Pharmacy with the approval of the Institutional Ethical Committee of GITAM deemed to be University, Visakhapatnam, India.

Experimental procedure:

Induction of diabetes:

Diabetes was induced in the rats by a single dosage of Streptozocin (STZ) (50mg/kg, i.p.) and they were also administered with 50% w/v sucrose solution. After 6 days of STZ injection the animals were subjected to cerebral ischemia- reperfusion injury, followed by collection of blood samples from the tail vein of rats for the estimation of glucose levels. The blood glucose levels of > 250mg/dl in rats were considered to be diabetic and such rats are included in the study. Spectrophotometrical estimation of serum glucose is employed by using commercially available kits (J. Mitra and Co. Ltd. New Delhi, India).

Experimental Induction Cerebral Ischemia:

Cerebral infarction was induced by bilateral common carotid artery occlusion method described by Orsu et al. 2013¹⁰. Briefly, rats were anesthetized with thiopental sodium dose 30mg/kg during surgical operation. Cervical vertebrae and the common carotid arteries were then exposed carefully and separated from the vagus nerve. These arteries were occluded for 30 min followed by reperfusion for 4 hrs. The rectal temperature was maintained at 37±0.5°C with a feedback-controlled heating-pad. Animals which convulsed during the ischemic episode were excluded.

Measurement of percentage cerebral infarct volume:

Chrysin was dissolved in 1% DMSO and administered intraperitoneally 5 min before reperfusion. Diabetic Rats were randomly divided into groups: sham, I/R (Ischemia-reperfusion), I/R+ vehicle and I/R+CH (Chrysin treated) (10, 20, 30, 40Hmg/kg). Each group consists of 6 animals. The coronal sections of 2 mm thickness were taken from the quickly removed brains after the predetermined time point of ischemia-reperfusion. Each slice was immersed in a 1.0% solution of 2, 3, 5-triphenyltetrazolium chloride (TTC) for 30min. Pale necrotic infarcted tissue and healthy, normal tissue stained dark red were separated and weighed. The percentage infarction was calculated¹⁰.

Estimation of cerebral oxidative stress biomarkers:

Oxidative stress biomarkers such as reduced glutathione (GSH), malondialdehye (MDA), xanthine oxidase (XO) and nicotinamide adenine dinucleotide phosphate (NADPH) levels were measured in the brain tissue. Glutathione Assay Kit was used to measure the levels of GSH spectrophotometrically at 412 nm using the colorimetric¹¹. The levels of MDA were assessed based on the reactivity of thiobarbituric acid (TBA) with malondialdehye (MDA) in the presence of acetic acid to yield MDA-TBA adduct, which was measured colorimetrically at 532nm¹². The levels of XO were assessed based on the reactivity of xanthine with 4-aminoantipyrine to generate a color. XO quantity was proportional to the color intensity, which was measured at 550 nm¹³. Double antibody sandwich technique was employed in the assay of NADPH ¹³, in which the NADPH quantity was proportional to the color intensity, which was measured at 450 nm. SOD¹⁴ and catalase¹⁵ were measured at 560 and 240 nm using spectrophotometrically.

Measurement of cerebral inflammatory biomarkers:

Myeloperoxidase¹⁰, tumor necrosis factor- alpha (TNF-α)¹⁶, interleukin-6 (IL-6)¹⁷, interleukin -10(IL-10)¹⁶ were measured using ELISA kits in which monoclonal antibodies used specific for rat respectively.

Determination of cerebral apoptotic mediators:

B-cell lymphoma 2 (Bcl2) protein¹⁸, Bcl2-associated X protein (BAX)¹⁹, and the heat shock protein 90 (Hsp90)¹⁸ were estimated by using ELISA kits in which monoclonal antibodies used specific for rat respectively.

Measurement of cerebral excitatory neurotransmitters:

Aspartate and the glutamate are excitatory neurotransmitters in the brain. The aspartate and glutamate levels were evaluated in the brain tissue by the using ELISA Kits^20,21.

Statistical analysis:

All values were expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Tukeys t test (P<0.05) using prism software 5.0.

RESULTS:

Effect of Chrysin on percentage of cerebral infarction:

There was a significant increase in percent cerebral infarction in I/R diabetic group compared to sham control group. A significant reduction in percent cerebral infarction was observed with Chrysin administration. Chrysin produced dose dependent effect by reducing cerebral infarction. Results were shown in Table 1.

Table No. 1 Effect of Chrysin on percentage cerebral infarction in diabetic rats

Groups (n=6)	Percentage cerebral Infarction
Normal	0
Sham control	5.61± 0.72
I/R	49.16± 0.34^*
Vehicle treated	49.22± 0.50
Chrysin (10 mg/kg, i.p.)	38.10± 0.28^*
Chrysin (20 mg/kg, i.p.)	29.41± 1.02^*
Chrysin (30 mg/kg, i.p.)	14.24± 0.15^*
Chrysin (40 mg/kg, i.p.)	9.72± 0.36^*

Data represent the mean±SEM, the asterisk indicates P≤0.05, statistically significant difference from control group, I/R indicates ischemia and reperfusion. No of animals used in each group =6

Effect of Chrysin on cerebral oxidative stress biomarkers:

MDA, XO, NADPH levels were significantly increased and Glutathione, SOD, CAT levels were significantly decreased in I/R group of diabetic rats as compared to sham control group. In Chrysin treated group, MDA, XO, NADPH levels were significantly reduced and Glutathione, SOD and CAT levels were increased significantly. Results were shown in Table 2.

Effect of Chrysin on cerebral inflammatory markers:

TNF-alpha, IL-6 and MPO levels were significantly increased and IL-10 levels were significantly decreased in I/R diabetic rats as compared to sham. In Chrysin treated diabetic rats, TNF-alpha, IL-6 and MPO levels were significantly reduced and IL-10 levels were significantly increased. Results were shown in Table 3.

Table No.4 Effect of Chrysin on Apoptotic levels

Assessments	Normal	Sham control	I/R	Vehicle Treated	*Chrysin(30 mg/kg, i.p.)* Treated
BCl2 (ng/gm of wet tissue)	26.23±0.125	25.423±0.352	11.83±0.659^*	10.87±0.860	33.69±1.201^*
BAX (ng/gm of tissue)	2.06±0.061	0.286±0.021	4.07±0.082^*	4.10±0.054	1.093±0.051^*
Hps90 (pg/gm of tissue)	28.01±1.623	28.09±2.006	52.42±1.691	52.64±1.069	24.52±1.469

Data represent the mean±SEM, the asterisk indicates P≤0.05, statistically significant difference from control group, I/R indicates ischemia and reperfusion. No of animals used in each group =6

Table No.5 Effect of Chrysin on cerebral Excitory neurotransmitters markers in diabetic rats

Assessments	Normal	Sham control	I/R	Vehicle Treated	*Chrysin(30 mg/kg, i.p.)* Treated
Glutamate (mg/gm of wet tissue)	3.603±0.205	5.632±0.425	96.94±0.748^*	97.02±0.910	4.96±1.910^*
Aspartate (nmol/g of tissue)	0.206±0.006	0.286±0.081	0.407±0.012^*	0.410±0.036	0.093±0.080^*

Data represent the mean±SEM, the asterisk indicates P≤0.05, statistically significant difference from control group, I/R indicates ischemia and reperfusion. No of animals used in each group =6

DISCUSSION:

The present study was involved to investigate the possible cerebroprotective mechanisms of Chrysin against ischemia-reperfusion injury in diabetic rats. The clinical outcomes were more worsened in diabetic associated ischemic stroke. Both diabetes and ischemia-reperfusion involve in over release of ROS, inflammation and also decline in levels of glutamate and aspartate. This can lead to further serious pathological events in the ischemic tissue injury. Few studies have revealed that anti-inflammatory and anti oxidant agents may be useful in limiting the reperfusion injury^22,23. Recently Chrysin showed neuroprotective role in rats^24,25,26. Recently researchers also suggested that common carotid arteries occlusion (CCA) can induce brain ischemia in rats¹⁰. CCA occlusion and reperfusion is followed by the pathological events such as inflammation and free radicals generation to cause tissue apoptosis in diabetic rats 27. Chrysin may be used in wide variety of conditions including cancer, cardiovascular and neurological disorders^6,7,8,24. Chrysin exerts multiple biological effects, including anti convulsant, antioxidant, anti-inflammatory, antihypertensive, antidiabetic and antiviral effects^6,7,8,24,28. Flavonoids were thought to be having beneficial health promoting properties in people of modern day life style. Chrsyin have the ability to show the antioxidant and anti-inflammatory effects like many other flavonoids. There were studies reporting the anti inflammatory effects of chrysin by inhibition of nuclear transcription factor ĸB (NK-ĸB), IL-1β, IL-2, IL-6, TNF-α and interferon-γ. Chrysin was thought to be an antagonist of NF-ĸB and also as an agonist of PPAR-γ (peroxisome proliferator-activated receptor gamma). By acting as agonist of PPAR-γ, chrysin downregulate the synthesis of proinflammatory ezymes like cyclooxegenase-2, MPO, inducible nitric oxide synthase (iNOS) and also phospholipase A2. By these collective effects chrysin can have its activity in improving neurodegenerative diseases²⁹. Estimating of cerebral damage can be done by evaluating the infarction area. By staining the coronal sections of brain with TTC can determine the size of infarct. The viable cells appear in deep red color and the infarct tissue as pale whitish/ unstained. We have noticed a significant increase in percentage of infarction among I/R diabetic rats and decreased percentage (dose dependent) among chrysin treated rats. These results were in accordance with the earlier reports¹⁰. These results of limiting the cerebral infarction suggest the cerebroprotective properties of Chrysin.

The ischemia/reperfusion injury can increase the basal oxidative stress in diabetic rats³⁰. Lipid peroxidation generates reactive aldehydes like malondialdehyde (MDA) which forms adducts with macromolecules and mimic the reactive oxygen species effects³¹. The first line antioxidants in the body include SOD, CAT and glutathione peroxidase which help in scavenging of free radicals in the body. In oxidative stress condition there will be an imbalance between free radicals and antioxidants³². The oxidative stress was found to be greater in ischemic rats when compared with the normal rats. This study also identified the reduction of antioxidants like glutathione, CAT and SOD with increase in oxidative markers like MDA, NADPH and XO among I/R diabetic rats. Whereas, in chrysin treated rats there was a significant increase in glutathione, CAT and SOD with decrease in MDA, NADPH and XO. These results show the antioxidant and reduced lipid peroxidation effects of the chrysin. In support to this several studies have demonstrated the antioxidant, anti-inflammatory properties of chrysin³³. Through these results we suggest the cerebroprotective action of chrysin against cerebral ischemia reperfusion injury by the antioxidant property. Post ischemic inflammation can be a consequence of cerebral infarction and neuronal injury. The endogenous mediators like cytokines, chemokines and leucocytes are involved in the inflammation and accumulate during the injury in brain tissue. The inflammatory mediators are initiated by cytokines like TNF-α, IL-1β and IL-6 which also involve in expression of other cytokines^34,35. The cytokine IL-10 is an anti-inflammatory agent which is expressed in response to the brain damage. It has activity against IL-1 and TNF-α and is supported by many studies³⁶. In this study we have noticed significant decrease in IL-10 and increase in MPO, TNF-α and IL-6 among I/R rats. Whereas, the levels of IL-10 has increased significantly and MPO, TNF-α and IL-6 levels were decreased among chrysin treated rats. This suggests the anti-inflammatory effects of chrysin in ischemia reperfusion injury.

Recent findings shows that Chrysin has a effective cardioprotection against doxorubicin-induced acute cardiotoxicity in rats through suppressing Inflammation, oxidative stress, and apoptotic tissue damage, via waning Casapase-3, BAX and cytochrome c expressions while increasing the expression of Bcl2³⁷. In the present study, we noticed remarkable declining levels of Bcl2 and increased levels of BAX and Hsp90 in I/R treated diabetic rats. However, Bcl2 levels were significantly increased and BAX, Hsp90 levels were significantly reduced in Chrysin treated diabetic rats. The level of excitatory neurotransmitters, aspartate and glutamate were increased through the administration of chrysin in comparison to I/R group. Considerable evidence shows that flavonoids have specific effects on the glutamate system and that this contributes to their ability to reduce excitotoxicity³⁸.

CONCLUSION:

The present study results constituted the evidence that the chrysin has significant cerebroprotective activity by reducing percentage of infarction. In diabetic rats chrysin has showed inhibitory effects against oxidative stress (MDA, XO NADPH), inflammation (MPO, TNF-alpha, IL-6 and IL-10) and apoptosis (Bcl2, BAX, Hsp90) caused by cerebral ischemia and reperfusion injury. This study suggests the anti oxidant, anti inflammatory and anti apoptotic effects of chrysin are involved in the cerebroprotection against Ischemi-reperfusion injury and further supports the possible use of chrysin as a therapeutic agent to ameliorate cerebral infarction.

COMPLIANCE WITH ETHICAL STANDARDS:

This study was approved by the animal Institutional ethical committee of GITAM Deemed to be University, India.

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest.

Source of support: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Received on 26.11.2019 Modified on 11.03.2020

Research J. Pharm. and Tech. 2021; 14(4):2049-2054.

DOI: 10.52711/0974-360X.2021.00364

Assessments	Normal	Sham control	I/R	Vehicle Treated	*Chrysin (30mg/kg, i.p.)* Treated
MDA (nMol/gm of wet tissue)	136.6±0.29	146.62±0.97	658.62±3.41^*	662.93±3.46	139.96±3.26^*
SOD (Units/mg of protein)	12.86±0.12	11.18±0.36	4.97±0.42^*	4.96± 0.52	10.12±0.42^*
CAT (Units/mg of protein)	119.06±2.12	113.71±3.25	34.39±1.36^*	34.28± 1.92	116.29±2.16^*
GSH (nmol/mL tissue)	2.12±0.02	2.02±0.09	1.02±0.05	1.13±0.12	2.26±0.16
XO (nmol/mg tissue)	10.23±0.98	10.12±0.90	29.32±1.02	29.65±1.92	9.96±1.84
NADPH (nmol/mg tissue)	12.36±1.20	12.69±1.09	30.02±1.66	30.96±1.90	11.92±1.31

Assessments	Normal	Sham control	I/R	Vehicle Treated	*Chrysin(30mg/kg, i.p.)* Treated
MPO (Units/gm of wet tissue)	3.603±0.205	5.632±0.425	96.94±0.748^*	97.02± 0.910	4.96±1.910^*
TNF-α (ng/mg of tissue)	0.206±0.006	0.286±0.081	0.407±0.012^*	0.410±0.036	0.093±0.080^*
IL-6 (ng/mg of tissue)	0.336±0.042	0.342±0.051	0.809±0.056^*	0.810±0.066	0.297±0.028^*
IL-10 (ng/mg of tissue)	3.080±0.064	2.672±0.063	0.895±0.068^*	0.870±0.043	1.976±0.23^*