Transanethole protects against Global Cerebral Ischemia through its Anti-inflammatory and Anti apoptotic activity

 

Hima Saila. M1*, Santhrani Thakur2

1Assistant Professor, Department of Pharmacology, Sri Padmavathi School of Pharmacy,

Tiruchanur, Tirupati, Chittoor Dist, Andhra Pradesh, India - 517503.

2Department of Pharmacology, Institute of Pharmaceutical Technology,

Sri Padmavathi Mahila Visvavidyalayam, Tirupati, Chittoor Dist, Andhra Pradesh, India-517502.

*Corresponding Author E-mail: himasaila.spsp@gmail.com

 

ABSTRACT:

Aim and Objective: In this research study, transanethole at a dose of 250 and 500mg/kg p.o was investigated for its potency against Bilateral Common Carotid Artery occlusion (BCCAO) for 1 hr accompanied by 22 hrs reperfusion. Method: Healthy Albino Wistar rats (200–250gm) were divided randomly into 4 groups (n=9).  Group I was considered as sham control, received 2% tween 80p.o, group II was considered as ischemic- reperfusion (disease control) and received 2% tween 80p.o and group III and IV were considered as prophylactic treatment groups and received transanethole at doses of 250mg/kg, p.o and 500mg/kg, p.o. respectively. After pretreatment with transanethole for14 days, rats were subjected to bilateral common carotid artery occlusion (BCCAO) for 1 hour accompanied by 22 hr reperfusion (I/R). After 22 hrs of reperfusion, motor coordination, behavioral assessment, infarct area, brain water content, excitatory mediators, inflammatory and apoptotic markers were determined. Results: Transanethole improved the motor coordination, lowered the brain water content, infarction volume and attenuated the levels of excitatory mediators. Western blotting analysis was performed to identify the proinflammatory mediators (TNF α, p38 MAPK), anti-inflammatory cytokine IL 10 and apoptotic marker proteins (Caspase 3, Bcl-2 and Bax) in brain tissue. Prophylactic treatment with trans anethole significantly down regulated the expressions of TNF-α, caspase 3, Bax, dephosphorylated p38 MAPK and upregulated the expressions of Bcl-2, IL 10 in a dose dependent manner in comparison with disease control group. Conclusion: An anti-inflammatory and anti- apoptotic activity of transanethole protected from cerebral ischemia reperfusion injury.

 

KEYWORDS: Ischemic reperfusion, cerebral infarction, transanethole, western blotting, anti apoptotic activity, anti-inflammatory activity.

 

 


INTRODUCTION:

Cerebrovascular maladies are caused by drastic reduction in supply of the oxygen to the brain. The brain stroke is one of the most widely recognized reasons for mortality and morbidity around the world. Concurrent conditions associated with cerebral stroke include lack of speech, visual problems, mental issues and loss of muscle coordination. Patients who suffered with stroke end up with physical and functional disabilities1.

 

Timely prevention of the disease is challenging and management of ischemic acute stroke is still a difficult task2.

 

Stroke is the result of various mechanisms that damage brain tissue. But the designed drugs target one or two mechanisms making them limited to treat stroke effectively. Considering the multifactorial pathophysiology of stroke, the design and development of the drugs should also concentrate on multiple pathways. So there is an immediate requirement to synthesize drugs that prevent and treat effectively without causing side effects. Drugs derived from herbal sources are found to be effective in many ways and cause fewer side effects too. Transanethole is commonly found in volatile oils. The Chemical name is 1-Methoxy-4-[(1E)-prop-1-en-1-yl] benzene derived from phenylpropene. Major families that contain transanethole are Fabaceae, Schisandraceae, Iliaceae, Apiaceae, Myrtaceae3. It possess antioxidant, antimicrobial, antifungal, anti-inflammatory, anticancer, anti-rheumatic,diuretic and used in alzhemier’s disease4 and protects neuronal cells (in-vitro)5. It is also proven to have antipyretic, analgesic, hypnotic and anticonvulsant activity. This study was designed to assess the probable protective mechanisms of transanethole in cerebral injury caused due to the occlusion of Bilateral Common Carotid Artery (BCCA) in rats.

 

MATERIALS AND METHODS:

Experimental Animals:

Albino rats weighing 200-220g were procured from Biogen laboratory animal facility, Bangalore. They were maintained under standard laboratory conditions and allowed for acclimatization for 7 days. They were kept in polypropylene cages and allowed to have free access to standard pellet feed and water ad labitum. The study protocol for the experiment was approved by IAEC (No.1016/PO/Re/S/06/CPCSEA/2019/010). Handling of the animals was done by following CPCSEA guidelines.

 

Chemicals:

Transanethole was procured from Sigma Aldrich, Mumbai.India. 2, 3, 5 Triphenyltetrazolium chloride was brought from SD fine India. Antibodies of Tumor necrosis factor (TNF α), Bax, Caspase 3, Bcl-2, p38 MAPK, IL 10and Horseradish peroxidase (HRP) were purchased from AbCham Inc. Analytical grade chemicals were used for the experimental procedures.

 

Grouping and Dose administration:

Rats were divided into 4 groups each consisting of 9 animals.  First group was sham control group, given 2% tween 80 orally. Second group was disease control [Ischemia reperfusion (I/R)] which received 2% tween 80 orally. Third and fourth group animals were administered with transanethole at a dose of 250 and 500 mg/kg orally6,7 respectively. The vehicle and trans anethole administrations were carried out for 14days prior to ischemia reperfusion.

 

Experimental Protocol:

Induction of brain injury was performed by cerebral ischemia reperfusion method Iwasaki et al8. Animals were first anesthetized using Ketamine at 100mg/kg/IM and Xylazine at 10mg/kg/IM injections. Incisions were made on either sides and the common carotid arteries were exposed. Area between sternocleidomastoid muscle and sternohyoid muscle was dissected parallel to the trachea. Ischemia was induced by occluding the carotid arteries using sterilized cotton thread. Occlusion was released after 1hr and the blood flow was perfused. Surgical wounds were sutured and povidone iodine ointment was applied with sterile cotton over the sutured area. The rats were allowed for 22 hrs of reperfusion and were subjected to various tests to analyze the neurological outcome and were sacrificed later to know the size of infarct and water content of brain. Sham group animals were also subjected to the similar surgical treatment but without occluding the arteries.

 

Behavioral Assessment:

Motor coordination:

Beam walking test was evaluated to assess motor coordination. Individual rats were kept on a wooden beam suspended at an inclination of 60ş from the platform. The performance of animals was recorded by using a 4 point scale. Animals that easily cross the beam were given score 0; Animals that had mild, moderate and severe impairment were given scores as 1, 2 and 3 respectively. Animals showed a complete inability to walk were given score 49.

 

Locomotor activity:

The locomotor activity was carried out by using Actophotometer. Rats were habituated in the Actophotometer for 3min and analyzed for 5min. The ambulatory activity was recorded as photo beam count per 5 min10.

 

Hanging wire test:

The test was used to estimate the forelimb grip strength of the animals after reperfusion. A wire was stretched between the edges and a foam pillow was placed to avoid injury. The rats were suspended by the forelimbs. The time taken for the animal to fall down was recorded and the experiment cut off time was set as 90 sec11.

 

Despair swim test:

Rats were placed in a cylinder with water where the rats could not touch the bottom with limbs or tail. The rats were not allowed to climb up the chamber too. Initially 15 min pretest swimming sessions were conducted followed by a 5 min session after 24 hrs. The period of immobility was noted (floating on water without struggling) for 5 min12,13.

 

Measurement of infarct area& water content:

Animals were anesthetized using Ketamine at 100mg/kg/I.M and Xylazine at 10mg/kg/I.M injections. They were sacrificed by decapitation and the brains were isolated. They were stored at 40C in normal saline and were analyzed for brain water content and infarct area measurement. Brains were sliced into 2mm thick sections and were immersed in 2% solution of 2,3,5-triphenyltetrazolium chloride. These slices were photographed. The brain infarction area was assessed by image J analysis software14,15.

 

Biochemical Estimation:

Brain homogenization:

After 22 hours of reperfusion, the animals were sacrificed by decapitation after inducing anesthesia. The brain was isolated. Brain tissue was homogenized in 5mM phosphate buffer containing 0.1mM EDTA to yield 10% w/v homogenate. This homogenate was subjected to centrifugation at 10,000rpm for 15 min and the supernatant was separated and stored in refrigerator at- 5şC and is used for biochemical estimation16.

 

Total Calcium level:

Calcium levels were estimated by using the commercially available calcium estimating kits (Span diagnostic Ltd., India).

 

Glutamate levels:                                                                                                                                                           

1ml of the tissue supernatant was mixed with 2ml of perchloric acid and pH was adjusted to 9 with phosphate buffer. This mixture was centrifuged at 1500rpm for 15min and kept aside for 10 min. The mixture was filtered and absorbance was measured at 340nm17.

 

Sodium-potassium ATPase (Na+-K+ATPase) activity:

A reaction mixture was prepared using 0.5ml 0.2M potassium chloride, 0.5ml of 1M sodium chloride, 0.5 ml of 0.2M Tris HCl buffer, 0.5ml of 0.1M magnesium chloride and 0.2ml of the supernatant to make a final volume of 2.2ml. Another mixture was prepared by mixing 0.5ml of 0.1M magnesium chloride, 0.5ml of 0.2 M Tris HCl buffer, 0.5ml of 0.1M sodium chloride and 0.5ml of 10mM of Oubain and 0.2ml of homogenate supernatant to make a final volume of 2.2ml. 0.4ml of 25mM ATP solution was added to both the mixtures at the same time and the temperature was maintained at 37 şC. They were allowed to react for 15 min. 0.1ml of 10% Trichloroacetic acid was added to the mixtures to stop further reaction. This was chilled and centrifuged at 1000rpm for 5 min and the supernatant was collected to estimate the phosphorus by following the method of Fiske and Subbarow18.

 

Western blot analysis:

The isolated brain tissue was homogenized by using  Radio Immuno Precipitation (RIPA) buffer [50mMTris-HCL pH 8.0, 150mM NaCl, 1% Nonidet P-40(NP40) or 0.1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% Sodium dodecyl Sulphate (SDS), 1mM Sodium orthovanadate, 1mM NaF, Protease inhibitors 10µl/ml]. This solution was heated at 70°C for 5 min and was used for separation of proteins using electrophoresis on 4% SDS-PAGE gel. 6μL of TrisHCl loading gel buffer and 15μL of the samples were loaded in each well and allowed to run for 1hr. The proteins were transferred on to the Polyvinylidene Flouride (PDVF) membrane and this membrane was stained using Ponceau stain to check the quality of transfer.

 

Blocking was performed in 3% skim milk in 20mM Tris buffer 150mM saline with 0.1% Tween 20 (TBST) in 1XPBS for 1hr. It was washed for 3 times using PBST solution for 5 min while maintaining constant temperature. Incubation was carried out with primary antibody (1:1000 of TNF α, p38MAPK, IL10, Bax, caspase 3 and Bcl-2) in 1XPBS overnight with continuous shaking at 4°C. Membranes were further incubated with horseradish peroxidase conjugated secondary antibody for about 2hrs and washed with 1XPBST thrice for 5min. This membrane was visualized using chromomeric substrate- 3, 3’-diaminobenzidine (DAB). Visible bands were photographed and quantitative densitometry analysis was done by image J analysis software. β-actin is chosen as loading control as it’s a housekeeping protein seen in almost all eukaryotic cells.

 

Statistical Analysis:

The values were expressed as mean and their corresponding standard error of means (Mean±SEM). Comparison was performed by using one way ANOVA and the significance of differences between groups was determined following Dunnet’s analysis. P<0.05 was considered as significant.

 

RESULTS:

Effect of transanethole Motor coordination and behavioral outcome:

The rats were analyzed for the motor coordination after 22 hrs of reperfusion and were scored on a 4 point scale. Data given in the table 1 represents that disease control group showed significant decrease in motor coordination in comparison with sham control. Transanethole showed a significant improvement in the motor coordination compared to disease control in dose dependent manner.

 

The behavioral outcome was assessed by observing locomotion, grip strength and immobilization (despair swim test). Disease control animals exhibited significant diminishment in locomotion, grip strength and mobility compared to sham group. Prophylactic treatment with transanethole at doses 250mg/kg and 500mg/kg exhibited remarkable improvement in locomotion, grip strength and mobilization compared to disease control group as shown in table 1.

 

Table 1: Effect of transanethole on motor coordination and behavioral outcome after Ischaemic reperfusion in rats

Treatment group

Motor co-ordination

Locomotor activity (count)

Hanging wire test (sec)

Immobility (sec)

Sham control

0.00

202.3±8.652

89.79±0.58

41.52± 8.61

Disease control (I/R control)

3.34±0.67+

94.6±7.325+

24.86±0.28+

92.65± 7.51+

Trans anethole (250 mg/Kg p.o)

2.51±0.41***

150.4±2.845***

69.34±0.41***

66.92±9.67***

Trans anethole (500 mg/Kg p.o)

1.28± 0.16***

187.6± 2.957**

55.41± 0.61***

48.57± 8.23***

All values were expressed as mean ±SEM (n=9) +p< 0.05 compared to sham control, ***p<0.001 compared to disease control (I/R control).

 


Effect of transanethole on Brain water content and infarction:

Figure 1 shows the brain water content and % infarction after 22 hrs of reperfusion. The results revealed that there was a noticeable increase in the water content of brain and infarction area in disease control compared to the sham group. This was reversed in the groups treated with transanethole 250mg/kg and 500mg/kg and found to be significant compared to disease control. The brain water content and % infarctions of all the groups were calculated and represented in table 2 and was shown in figure 1.

 

Table 2: Effect of transanethole on the infarct area and brain water content:

Treatment group

% infarction

Brain water content

Sham control

--

0.034±0.024

Disease control (I/R control)

71.64***

4.48±0.584***

Trans anethole (250 mg/Kg p.o)

22.18+++

2.24±0.560+++

Trans anethole (500 mg/Kg p.o)

10.47+++

1.04±0.454+++

All values were expressed as mean±SEM (n=9) ***p< 0.001 compared to sham control, +++p<0.001 compared to disease control (I/R control).

 

A. Sham control B. Disease control (Ischaemic control) C. trans anethole 250 mg/kg D.transanethole 500 mg/kg

Fig 1: Pictures representing the TTC stained coronal sections of brain

 

Effect of trans anethole on excitatory mediators and Na+k+ATPase activity:

The results in table 3 showed significant increase in calcium and glutamate levels in disease control group in comparison with sham control. These levels were significantly reversed by the prophylactic treatment with transanethole in dose dependent manner in comparison with disease control. There was significant diminishment in Na+K+ATPase levels in disease control group compared to sham control. Prophylactic treatment with transanethole showed significant enhancement of Na+-K+ATPase levels in dose dependent manner in comparison with disease control.

 

Table 3: Effect of transanethole on Calcium, Glutamate and Na+/K +ATPase after ischaemic reperfusion injury in rats

Treatment group

Calcium (µg/mg protein)

Glutamate (µg/mg protein)

Na+/K+ATPase (µM of pi liberated/hr/mg protein

Sham control

10.63± 1.16

8.10±0.89

8.68±0.89

Disease control (I/R)

35.73± 2.70***

25.92± 1.86***

2.43± 0.58***

Trans anethole (250 mg/Kg p.o)

29.28± 1.64+++

19.41± 0.78+++

4.18±0.74+++

Trans anethole (500mg/Kg p.o)

18.53± 1.32+++

12.01± 0.67+++

6.65±0.71+++

The values were represented as mean±SEM (n=9). +++P<0.001compared with disease control (I/R) group and ***P<0.001 compared with sham control group.

 

Western blotting analysis:

Western blotting was performed to analyze the presence of proinflammatory and anti inflammatory mediators, involvement of p38 MAPK and cell apoptosis mediators in the brain after 22 hrs of reperfusion.

 

Effect of transanethole on proinflammatory mediator TNF α:

As shown in figure 2 the expressions of proinflammatory mediator TNF α in disease control were up-regulated markedly compared to sham control and was found to be down-regulated markedly in prophylactic transanethole treated groups compared to disease control.

 

Fig.2 Western blotting analysis of TNFα protein expression in all experimental groups

The bar graphs represented relative expression of TNF α corresponding to β actin. Data was represented as mean±SEM (n=9). ++P<0.001 compared with disease control (I/R) group and ***P<0.001compared with sham control.

 

Effect of trans anethole on proinflammatory mediator p38 MAPK:

As shown in figure 3, Phosphorylation of p38 MAPK was observed in disease control and was found to be significant compared to sham control and dephosphorylation of p38 MAPK was noticed in prophylactic treated transanethole groups compared to disease control.

 

 

Fig 3 Western blotting analysis of p38 MAPK protein expression in Sham, disease control and trans anethole treated groups.       

The bar graphs represented relative expression of p38 MAPK corresponding to β actin. Data was represented as mean±SEM (n=9). ++P<0.001 compared with disease control (I/R) group and ***P<0.001 compared with sham control

 

Effect of trans anethole on anti-inflammatory mediator IL 10:

As shown in fig 4,IL 10 was released in response to brain reperfusion injury that was evidenced by the appearance of bands in the animals treated with transanethole and was found to be significant compared to disease group.

 

 

Fig.4 Western blotting analysis of  IL 10 protein expression in all experimental groups

The bar graphs represented relative expression of IL 10 corresponding to β actin. Data was represented as mean±SEM (n=9).++P<0.001 compared with disease control (I/R) group and nsP>0.05compared with sham control, ***P<0.001 compared with sham control.

 

Effect of transanethole on anti –apoptotic (BCl-2) and pro-apoptotic (Bax, Caspase 3) mediators:

As shown in figure 5, after reperfusion phase for 22 hrs, Disease control group presented considerable lowering in BCl-2 expression compared to sham group, at the same time prophylactic treatment with transanethole groups markedly showed the up-regulation in expression of BCl-2 compared to disease control. Reversing it, appearance of caspase 3 and Bax were upregulated clearly in disease control compared to sham control which was considerably declined by the prophylactic treatment with transanethole. 

 

Fig 5a

 

 

Fig 5b

 

Fig 5c

 

Fig 5(5a, 5b, 5c) : Western blotting analysis of Bcl-2, Bax and caspase 3 protein expression in all experimental treated groups.

The bar graphs represented relative expression of Bcl-2, Bax and caspase 3 corresponding to β actin. Data was represented as mean±SEM (n=9).++P<0.001 compared with disease control (I/R) group and ***P<0.001 compared with sham control.

 

DISCUSSION:

Stroke is one of the major causes of the death of patients and is caused due to peri-infarct depolarization, oxidative stress, ionic imbalance, excitotoxicity, inflammation, acidotoxicity and apoptosis. The major challenge in brain research is to develop newer methods of effectively managing the strokes which occur due to any of afore mentioned factors19. In this study transanethole was investigated for its activity to prevent the brain injury caused due to the BCCAO followed by reperfusion.

 

In this model, disease control group exhibited marked impairment in motor coordination and significant loss or impairment of the sensory and motor activities. This was evident from the poor performance of the rats in the locomotor activity, grip strength and despair swim test. Prophylactic transanethole treated groups exhibited significant enhancement in motor coordination, locomotion, grip strength and despair swim test in comparison with disease control. This suggests the involvement of transanethole in preventing the injury of brain caused by ischaemic reperfusion.

The size of infarction was assessed by TTC. As TTC is sensitive to NAD and NADHPH it converts into red formazone pigment seen in active cells. Due to lack of these enzymes the cells that are damaged appears to be pale and colourless. The infarction area was decreased significantly in the groups treated with transanethole in comparison with disease control. The brain edema content was significantly decreased in prophylactic treatment groups in comparison with disease control group.

 

During cerebral ischemia reperfusion injury failure of Sodium Potassium ATPase pumps cause accumulation of sodium ions together with hydrogen ions that cause inflammation of brain tissue. Besides this, due to energy depletion, depolarization of neuronal cell membranes takes place and cause to release more amount of glutamate that actsas an excitatory neurotransmitter in CNS. Overproduction of glutamate leads to excitotoxicity that causes further enhancement of calcium and imbalances the calcium homeostasis. Calcium ions play an important role that mediates cell functions like exocytosis, excitation, growth, differentiation and synaptic activity. Neurons have own unique homeostatic mechanisms to control calcium present in cytoplasm. Even though increase in calcium levels intracelluarly are observed in normal physiological conditions but during any pathological situations specifically in brain injury calcium also released from endoplasmic reticulum, this situation  devastate the management of  calcium regulation and leads to cell death20. Enhanced calcium and glutamate levels were remarkably increased in disease control groups that were reversed by prophylactic treatment with transanethole in dose dependent manner.

 

The main hallmark involved in response to reperfusion damage is activation of microglia and immune cells infiltration, a crucial patron of inflammation. These cells activated considerable and crucial mediators of the inflammatory cascade that are cytokines throughout the brain injury and perhaps produced in asymmetrical fashion that are deleterious to neurons. During reperfusion brain damage tumor necrosis factor αand IL-1β are primary cytokines that are going to be up-regulated. The generation and liberation of these effectors turn on inducible gene expression by cell signaling activation. Firstly TNF receptor (TNFR1 and TNFR2) gets stimulated by inflammatory signals that operate by stimulating the cell membrane receptors. These receptors activation leads to switch on the P38 Mitogen-Activated Protein Kinase (MAPK) pathway. In our research study, ischemia reperfusion injury associated with the activation of p38 MAPK cascade pathway is mainly mediated by proinflammatory cytokine TNF α and results in upregulation of genes of apoptosis.  Excitotoxicity, failure of energy and activation of microglial cells also leads to production of proinflammatory mediators and results in phosphorylation of p38 MAPK signaling cascade21,22. P38 MAPK is one of the major among MAP kinases, which has been involved in management of physiological cell measures, including survival, proliferation, adaptation, apoptosis and inflammation. From our study it was evidenced that p38 MAPK cascade imparts signals from cytosol towards nucleus and turn on transcription genes by means of activating downstream kinases, harmonizes the expression of  genes correlated with apoptosis, thus promoting cellular apoptosis23,24. Disease control group remarkably showed upregulation of TNF α and phosphorylation of p38 MAPK that were markedly downregulated and dephosphorylated by the prophylactic treatment with transanethole.

 

On the other hand in response to microglial activation, anti-inflammatory mediator, IL 10 protein expression was observed in the transanethole treated groups in dose dependent manner in comparison with disease control. IL 10 plays a pivotal role in quenching the inflammatory response in central nervous system. IL 10 is capable to hinder the production of proinflammatory cytokines by microglia thus safe guarding the astrocytes from excessive inflammation. IL 10 receptor cascade has been correlated with improvement in cellular survival25.

 

According to the present study, depletion of energy, excess levels of calcium, excitotoxicity and activation of inflammatory cascade leads to dysfunction of mitochondria thereby increasing its permeability thus makes the cell to undergo apoptosis. The apoptosis is under the tight control of pro-apoptotic genes (BAX, Bad) and anti-apoptotic genes of BCl-2 family. Due to activation of intrinsic pathway, the mitochondria releases cytochrome C that triggers the initiator caspases (caspase 9) then activates executioner caspases (Caspase-3). Activation of Caspase 3 makes the cell to undergo shrinkage, chromosomal DNA fragmentation, breakdown of nuclear material and cytoskeleton proteins 26. Prophylactic treatment with trans anethole remarkably showed down regulation of   Caspase 3 and Bax  ((pro-apoptotic mediators) and  up regulation of BCl-2 in comparison with disease control. Our research evidence indicated that prophylactic treatment with transanethole ameliorated the damage caused by cerebral ischemia reperfusion injury in dose dependent manner perhaps due to its anti-inflammatory and anti apoptotic potentiality.

 

CONCLUSION:

Based on the research evidences of the present study, it can be concluded that trans anethole prevented the brain damage in dose dependent manner by enhancing the motor coordination, behavioral outcome, by attenuating brain edema content, infarct volume, levels of excitatory mediators and down regulation of TNF alpha, Bax, Caspase-3 (pro-apoptotic mediators) and up-regulation of BCl-2, showed enhancement in the expression of anti-inflammatory cytokine IL10. These results suggested that prophylactic treatment of transanethole might be beneficial for use in cerebral ischemia injury. Further research is necessary to explore its role in humans.

 

ACKNOWLEDGEMENT:

Authors thank all those who supported the work and there was no funding support for carrying out this study.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 27.02.2021                Modified on 28.05.2021

Accepted on 20.07.2021               © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1713-1720.

DOI: 10.52711/0974-360X.2022.00287