INTRODUCTION:

Depression is a state of low mood and aversion to activity that can affect a person's thoughts, behaviour, feelings and physical well-being¹. It has been surveyed that by 2020 depression may become the second-most disabling disease after cardiovascular disease². Various investigations have been reported for the explorations of pathophysiology of depression^3-6.

Various herbal medicines have also been widely used across the world for their therapeutic effectiveness⁷. Despite growing evidences for such reports of investigations, there exist many limitations of current antidepressants treatment⁸ like suicide tendency and discontinuation syndrome and there is need of time to focus research on minimising side effects⁹.

Considering such reports of limitations of current antidepressant agents, it can be proposed that there is a need for the more exploration of pathophysiology of depression and also a need to develop antidepressant agents based on novel targets. Further, in the last few years, many studies have revealed the role of inflammation and inflammatory mediators in the neurobiology of depression^10-15.

Several reports indicated the abnormal prostaglandin E levels in depression^16-18. Various studies reported that there has been an elevated level of prostaglandins (PGs) especially PGE₂ in depression^{16-17, 19}. Further, it has been reported in various preclinical studies that PGE₂may contribute to neuro-inflammation from various preclinical studies²⁰.Several studies reported that depressed individuals may have increased levels of prostaglandin E₂ (PGE₂) in the saliva, plasma and CSF^21-22. Signs of an inflammatory process, particularly high prostaglandin E₂ (PGE₂) levels, have been described in major depression²³.

Further, increased levels of PGE₂has been correlated with up-regulation of cyclooxegenase-2 (COX-2)²⁴. COX-2 inhibitors inhibit the PGE₂, suggesting a positive role in depression^25-26.It has been reported thatvarious antidepressant drugs could inhibit PGE₂synthesis including MAO-inhibitors^27-30 and tricyclic antidepressants³¹. Loxoprofen is an analgesic category of drug, having anti-inflammatory properties. Various reports in preclinical studies indicated that Loxoprofen remarkably decreases the PGE₂ level in brain regions³².

Considering the above mentioned evidences, reports and property of inhibition of PGE₂ of Loxoprofen, it can be hypothesized that Loxoprofen may affect brain serotonin, noradrenaline and dopamine levels. Hence, it has been proposed to study the effect of Loxoprofen in Lipopolysaccharide induced alterations in serotonin, noradrenaline and dopamine levels in mice after chronic mild stress treatment.

MATERIAL AND METHODS:

Animals:

Swiss albino male mice were obtained from Zydus Research Centre, Moraiya, Ahmedabad. They were housed under standard conditions with free access to food and water, under 12:12 hr light: dark cycle. Mice were allowed to acclimatize for 07 days before the initiation of behavioural tests were performed. Each animal was used only once (N= 6 animals per group). The experiments were performed after the protocol for experimental design was approved with protocol no. BIP/IAEC/2018/06 by the Institutional Animal Ethics Committee (IAEC) of Babaria Institute of Pharmacy. The experiments were conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA).

Drugs:

Venlafaxine [VEN] as triple monoamine reuptake inhibitor was administered in the dose of 4mg/kg i.p.³³.Lipopolysaccharide [LPS] was administered in the dose of 0.5mg/kg, i.p³⁴Loxoprofen [LOX] was dissolved in 0.5% CMC solution³².Venlafaxine was dissolved in normal saline solution (0.9% NaCl)³³. LPS was dissolved in phosphate buffered saline (PBS) solution (pH 7.4)³⁴.

Chronic Mild Stress (CMS):

Stress schedule followed in paradigm:

Total sixty mice with ten groups were subjected to stress schedule. The stress schedule for producing chronic mild stress was followed according to the already reported stressor schedule ³⁵.The stress scheme included various stressors which included three five hour periods of water and food deprivation immediately before the sucrose tests, two periods of intermittent illumination, two periods (7 and 12 hr) of 45 degree cage tilting, one additional 16-hr period of water deprivation, one 12hr period in a soiled cage (adding 100ml water in the bedding) and three periods (7, 9 and 12hr) of low intensity stroboscopic illumination (150flashes/min). These stressors were scheduled every day for a total period of 21 days.

Groups of mice:

Out of these sixty mice, eighteen mice were housed under normal condition. They were subdivided into three groups as follows: Group I received saline and serve as vehicle control- I (VC-I), Group II received 0.5% CMC serve as vehicle control- II (VC-II), Group III received Lipopolysaccharide (0.5mg/kg, i.p.) (LPS). Rest of forty two mice were housed under chronic mild stress and were divided as follows: Group IV received stress and saline (10ml/kg, p.o.) (STR-I), Group V received stress and 0.5% CMC (10ml/kg, p.o.) (STR-II), Group VI received stress and Venlafaxine (4mg/kg, i.p.), Group VII received stress and Loxoprofen (16.8mg/kg, p.o.), Group VIII received stress and Lipopolysaccharide (0.5mg/kg, i.p.), Group IX received stress and Lipopolysaccharide (0.5mg/kg, i.p.) and Venlafaxine (4 mg/kg, i.p.), Group X received stress and Lipopolysaccharide (0.5mg/kg, i.p.) and Loxoprofen (16.8mg/kg, p.o.)_.

Measurement of brain serotonin, noradrenaline and dopamine levels in mice brain:

All mice of group one to group ten as mentioned above were sacrificed and their brains were removed. The isolated brains were kept frozen at - 80°C until assay. Whole brain of each mouse was weighed without thawing and immediately homogenized in 5mL ice cold acidified butanol. The homogenate was centrifuged at 500rpm at 4°C for 10 min and the supernatant was collected. Brain serotonin, noradrenaline and dopamine levels were estimated by flourometric method of Jacobowitz and Richardson³⁶.

Statistical analysis:

All quantified data were expressed as mean±S.E.M. for the indicated statistical analysis. Statistical comparisons were performed by one-way ANOVA followed by the Tukey’s Test. P<0.05 were considered as degree of significance. All statistical analysis was performed using approved statistical software (Sigmastat software, Systat Software Inc, San Jose, CA, USA.).

RESULTS:

Effect of Loxoprofen and Venlafexine on serotonin levels in mice brain:

There was a significant decrease in brain serotonin levels in mice of stress control-I and stress control-II as compared to vehicle control-I and vehicle control-II respectively. There was a significant decrease in brain serotonin levels in LPS treated stressed mice as compared to LPS treated normal mice. Furthermore, Venlafexine showed a significant increase in brain serotonin levels in stressed mice as compared to stress control-I while Loxoprofen showed a non-significant increase in brain serotonin levels in stressed mice as compared to stress control-II. However, the same treatment of Loxoprofen in LPS treated stressed mice showed a significant increase in brain serotonin levels as compared to LPS treated stressed mice. Similarly, the treatment of Venlafaxine in LPS treated stressed mice showed a significant increase in brain serotonin levels as compared to LPS treated stressed mice. (Table 1)

Effect of Loxoprofen and Venlafexine on noradenaline levels in mice brain:

There was a significant decrease in brain noradrenaline levels in mice of stress control-I and stress control - II as compared to vehicle control-I and vehicle control-II respectively. There was a significant decrease in brain noradrenaline levels in LPS treated stressed mice as compared to LPS treated normal mice. Furthermore, Venlafexine showed a significant increase in brain noradrenaline levels in stressed mice as compared to stress control-I while Loxoprofen showed a non-significant increase in brain noradrenaline levels in stressed mice as compared to stress control-II. However, the same treatment of Loxoprofen in LPS treated stressed mice showed a significant increase in brain noradrenaline levels as compared to LPS treated stressed mice. Similarly, the treatment of Venlafaxine in LPS treated stressed mice showed a significant increase in brain noradrenaline levels as compared to LPS treated stressed mice. (Table 2)

Table 1: - Effect of Loxoprofen and Venlafexine on serotonin levels in mice brain

Group No	Treatment	Dose (mg/kg)	Route	5-HT (ng/gm of brain tissue) [Mean ± SEM]
I	Vehicle Control-I	10 ml/kg	p.o.	167.73 ± 16.60
II	Vehicle Control-II	10ml/kg	p.o.	162.15 ± 24.16
III	Lipopolysaccharide	0.5 mg/kg	i.p	89.99 ± 6.84
IV	Stress Control-I	10 ml/kg	p.o.	112.28 ± 11.45^*
V	Stress Control-II	10ml/kg	p.o.	93.16 ± 14.26⁺
VI	Stress and Venlafexine	4mg/kg	i.p.	155.43 ± 11.55^$
VII	Stress and Loxoprofen	16.8 mg/kg	p.o.	102.58 ± 14.05
VIII	Stress and Lipopolysaccharide	0.5 mg/kg	i.p.	67.88 ± 5.74^#
IX	Stress, Lipopolysaccharide and Venlafaxine	0.5 mg/kg + 4mg/kg	i.p. & i.p.	219.21 ± 43.51^£
X	Stress, Lipopolysaccharide and Loxoprofen	0.5 mg/kg + 4mg/kg	i.p. & p.o.	102.59 ± 14.05^£

Each column expressed as Mean ± SEM of six animals after respective treatments. Data were analysed by One-way Analysis variance (ANNOVA) followed by Tukey’s test. *p<0.05 when compared with VC-I, +p<0.05 when compared with VC-II, #p<0.05 when compared with LPS, $p<0.05 when compared with STR-I, £p<0.05 when compared with STR+LPS.

Table 2 - Effect of Loxoprofen and Venlafexine on noradrenaline levels in mice brain

Group No	Treatment	Dose (mg/kg)	Route	NE (ng/gm of brain tissue) [Mean ± SEM]
I	Vehicle Control-I	10 mg/kg	p.o.	719.94 ± 73.75
II	Vehicle Control-I	10ml/kg	p.o.	768.06 ± 82.03
III	Lipopolysaccharide	0.5 mg/kg	i.p	619.59 ± 30.66
IV	Stress Control-I	10 mg/kg	p.o.	474.49 ± 50.66^*
V	Stress Control-II	10ml/kg	p.o.	510.82 ± 59.95⁺
VI	Stress and Venlafexine	1 mg/kg	i.p.	640.99 ± 50.83^$
VII	Stress and Loxoprofen	16.8 mg/kg	p.o.	527.88 ± 56.89
VIII	Stress and Lipopolysaccharide	0.5 mg/kg	i.p.	390.33 ± 76.92^#
IX	Stress, Lipopolysaccharide and Venlafaxine	0.5 mg/kg + 4mg/kg	i.p. & p.o.	659.22 ± 47.64^£
X	Stress, Lipopolysaccharide and Loxoprofen	0.5 mg/kg + 4mg/kg	i.p. & p.o.	611.61 ± 62.39^£

Each column expressed as Mean ± SEM of six animals after respective treatments. Data were analysed by One-way Analysis variance (ANNOVA) followed by Tukey’s test. *p<0.05 when compared with VC-I, ⁺p<0.05 when compared with VC-II, ^#p<0.05 when compared with LPS, ^$p<0.05 when compared with STR-I, ^£p<0.05 when compared with STR+LPS.

Table 3: - Effect of Loxoprofen and Venlafexine on dopamine levels in mice brain

Group No	Treatment	Dose (mg/kg)	Route	DA (ng/gm of brain tissue) [Mean ± SEM]
I	Vehicle Control-I	10 mg/kg	p.o.	1602.96 ± 146.59
II	Vehicle Control-I	10ml/kg	p.o.	1357.12 ± 81.51
III	Lipopolysaccharide	0.5 mg/kg	i.p	1476.64 ± 117.17
IV	Stress Control-I	10 mg/kg	p.o.	1033.60 ± 38.70^*
V	Stress Control-II	10ml/kg	p.o.	1049.30 ± 97.52⁺
VI	Stress and Venlafexine	4mg/kg	i.p.	1482.32 ± 177.99^$
VII	Stress and Loxoprofen	16.8 mg/kg	p.o.	907.36 ± 193.19
VIII	Stress and Lipopolysaccharide	0.5 mg/kg	i.p.	790.44 ± 220.59^#
IX	Stress, Lipopolysaccharide and Venlafaxine	0.5 mg/kg + 4mg/kg	i.p. & i.p.	1378.86 ± 136.95^£
X	Stress, Lipopolysaccharide and Loxoprofen	0.5 mg/kg + 4mg/kg	i.p. & p.o.	1100.00 ± 350.82

Each column expressed as Mean ± SEM of six animals after respective treatments. Data were analysed by One-way Analysis variance (ANNOVA) followed by Tukey’s test. *p<0.05 when compared with VC-I, ⁺p<0.05 when compared with VC-II, ^#p<0.05 when compared with LPS, ^$p<0.05 when compared with STR-I, ^£p<0.05 when compared with STR+LPS.

DISCUSSION:

The results of the present investigations indicated the antidepressant-like effect of Loxoprofen in mice. The link between stress and depression is not novel. It has been reported that stress has become the leading cause for the neurobiology of depression. Various models involving chronic stress exposure to negative stressful events can be considered the most effective in modelling depressive behaviour³⁷.

Various theories have been proposed regarding the causes of depression. Among these theories the lack of neurotransmitter levels such as noradrenaline or dopamine levels in the brain has received the most common focused area of research. The theory that depression is caused by decreased levels of noradrenaline and serotonin, received considerable support also³⁸.

The results of study indicated that Loxoprofen alter the serotonin and noradrenaline levels in mice but not the dopamine level. However, the exact mechanism by which Loxoprofen indicated a significant antidepressant- like action in a chronic mild stress model remains to be elucidated. Though, the previously reported inhibitory action of Loxoprofen on PGE₂synthesis may show potential role of PGE₂ in mediating the antidepressant effect of Loxoprofen in animal models of depression. It can be possible that alterations in the brain serotonin or noradrenaline levels induced by Loxoprofen at specified dose as mentioned in the present study may be achieved by the inhibition of PGE₂ in brain. It is also possible that such Loxoprofen induced PGE₂ inhibition may be responsible for the alterations in brain serotonin or noradrenaline levels.

Although we were unable to measure brain PGE₂ levels in the brain but further research work is suggested to examine if Loxoprofen induced alteration in brain PGE₂ levels may affect the brain serotonin or noradrenaline levels in mice brain. It has been also suggested for the researcher to measure the serotonin or noradrenaline levels in particular parts of the brain that are mainly affected in depression.

The data from the neurotransmitter levels after chronic mild stress shows the potential antidepressant-like action of Loxoprofen in mice, but further work is required for further exploration of the present investigations.

Regardless any previous reports or studies, these are the first results for the potential antidepressant-like effect of Loxoprofen by showing the alterations in the brain serotonin and noradrenaline levels in mice brain after chronic mild stress treatment, having its further potential implication in the pathophysiology and treatment of depression in future.

CONCLUSIONS:

The results of the present study indicated that Loxoprofen influence LPS induced alterations in serotonin levels and noradrenaline levels in chronic mild stress treated mice. It also indicated possible antidepressant-like effect of Loxoprofen in mice subjected to chronic mild stress, having its possible implication in future treatment of depression.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS:

The authors are grateful to the management team of Babaria Institute of Pharmacy, Vadodara, India for providing necessary facilities for this work. Also thankful to Zydus Research Centre, Ahmedabad for providing animals and all other pharmaceutical companies for providing gratis sample of drug for this research. They are also thankful to Anand Pharmacy College, Anand for providing facility for spectrofluorometric analysis

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Received on 02.03.2021 Modified on 11.05.2021

Research J. Pharm. and Tech. 2022; 15(5):2081-2086.

DOI: 10.52711/0974-360X.2022.00344