Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38

Introduction

The 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and the third edition of the International Classification of Sleep Disorders (ICSD-3) define insomnia as difficulty initiating or maintaining sleep at least 3 nights a week or more and lasting at least 3 months.¹ The prevalence in Europe and Scandinavia shows an increasing prevalence. British study of more than 20,000 adults saw an increase in the average prevalence of insomnia over a 15-year period from 35% in 1993 to 38.6% in 2007, while the prevalence of insomnia in the United States was about 27%.^1,2

Based on gender, insomnia is more common in women than in men, which is 50% greater in women than men. Old age, low economic status, hyperarousal conditions, and the presence of psychiatric or medical comorbidities are risk factors associated with insomnia.¹ The latest concept of insomnia is the disintegration of molecules that play a role in alternation of waking and sleeping rhythms in the brain. One of the molecules that play a role in the circadian rhythm of arousal is hypocretin/orexin and histamine, while those that play a role in sleep, such as γ-aminobutyric acid (GABA), adenosine, serotonin, melatonin, and prostaglandin D2 (PGD2).³

The binding of orexin to orexin receptor type 1 (OXR1) or orexin receptor type 2 (OXR2) stimulates the Gq or Gi subtypes, sequentially activating phospholipase C (PLC), phospholipase A (PLA), phospholipase D (PLD) or Adenyl cyclase (AC), causing an increase in cytosolic Ca²⁺ and subsequent response cascades. OA binds to OX1R and increases Ca²⁺ by activation of nonselective cation channels (NSCCs) (Figure 1).³

Figure 1. Orexin system signal pathway.³

There are 2 receptor subtypes for orexin that will activate 3 G-protein subtypes (Gq/11, Gi/10, and Gs). Activation of Orexin Receptor-1 (OXR1 or OA) and Orexin Receptor-2 (OXR2 or OB) receptors activates phospholipase c cascade (PLC-IP3/DAG). Orexin receptor signaling pathways phospholipase D (PLD)/phosphatidic acid (PA), phospholipase A (PLA)/arachidonic acid (AA), and mitogen-activated protein kinase cascade (MAPK). Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. Extracellular Signal-regulated Kinases or ERK1/2 and p38 are rapidly phosphorylated in response to OA and OB, mediated primarily by Gq and slightly the Gi pathway. Orexin binding and orexin receptors will activate intracellular calcium PKC-dependent or independent pathways. Orexin receptors, both OA and OB, also stimulated the activation of Akt kinase in the cortical nerves of hypoxic stress-stressed rats. In addition, orexin will cause a reaction on OB to activate Gi proteins causing inhibition of cAMP formation. Orexin also activates OA to stimulate cAMP synthesis. Orexin signaling rapidly activates the mTORC1 pathway triggered by the lysosomal v-ATPase pathway.⁴

Centella asiatica has a role in the signal work of the MAPK/ERK pathway. Studies on human breast cancer cells show that Asiatic acid (AA) in gotu kola plays a role in inducing inhibition of cancer cell growth through mediators such as MAPK, ERK1/2, and p38. Asiatic acid targets P13K/Akt/mTOR to prevent growth and induction of apoptosis in ovarian cancer cells. In cardiovascular disease AA has benefits with its ability to inhibit excessive pressure that induces cardiac hypertrophy by inhibition of phosphorylation of p38, MAPK, and ERK1/2 and decreases the production of TGF-β and NF-КB.^5,6

Currently, many animal models of sleep have been developed for sleep disorder research. The zebrafish (Danio rerio) currently exhibit vertebral sleep models that are important among mouse and invertebrate models. Zebrafish sleep mainly at night as in humans, unlike rats that have the habit of sleeping during the day. The main advantages of zebrafish as sleep model animals are the discovery of genes involved in sleep control, neuropeptide systems, transparent anatomical structures of the brain, and allow for neuropsychiatric disease models, as well as models for genetic and pharmacological screening in sleep disorders.⁷ Zebrafish brains also contain orexin, one of the molecules important for sleep regulation in mammalian brains. At present high-speed infrared video capture combined with computational image analysis has been used to quantitatively describe locomotor behavior and sleep posture in zebrafish larvae.³ The purpose of this study was to prove the effect of gotu kola extract (Centella asiatica) in improving the sleep activity of zebrafish larvae (Danio rerio) insomnia model, extending the total inactivity time (cumulative duration) and shortening the duration of first sleep (latency to first sleep) in light and dark phases through inhibition of orexin, ERK, p38 and Akt.

Methods

Results

Average western blot analysis of orexin protein expression

Protein expression of orexin was measured in zebrafish larvae samples using the western blot method and gel doc chemiluminescence. The average results of orexin protein WB analysis are presented in the form of tables and diagrams as follows (Table 1 and Figure 2).

Table 1. Average Western blot analysis of orexin protein expression.

No.	Treatment	Number of samples (n)	Western blot protein expression of orexin ± SD (μg/μL)	p-value
1.	Normal Group (N)	144	122716.5 ± 5014.5b,c	<0.001
2.	Insomnia Group (I)	144	131239 ± 7994c
3.	I + 2.5 μg/mL CA	144	100565 ± 9707a,b
4.	I + 5 μg/mL CA	144	91963 ± 9129a
5.	I + 10 μg/mL CA	144	92655 ± 9604a

Figure 2. Diagram of the average result of WB analysis of orexin protein expression.

• 1. Normal Group (N) = kontrol negatif

• 2. Insomnia Group (I) = induksi cahaya

• 3. I + 2.5 μg/mL CA

• 4. I + 5 μg/mL CA

• 5. I + 10 μg/mL CA

• 6. Marker (Orexin)

Based on the average results of WB analysis of orexin protein expression, the insomnia group has a higher average expression value of 131239 ± 7994 μg/μL compared to the normal group (122716.5 ± 5014.5 μg/μL) and different concentrations of CA extract which are respectively 2.5 μg/mL (100565 ± 9707 μg/μL), 5 μg/mL (91963 ± 9129 μg/μL) and 10 μg/mL (92655 ± 9604 μg/μL). Various concentrations of CA extract can reduce the average expression value of orexin protein which basically functions as a wake-up trigger in the sleep-wake cycle. Statistical analysis also showed that there was a significant difference in orexin protein expression values, this was evidenced by significance signs in the form of (^a,b,c) which showed that exposure treatment of CA extract was proven to reduce orexin protein expression values significantly lower than normal and insomnia groups. The location of orexin protein is at 49 kDa (Table 1 and Figure 2).

Average Western blot analysis of ERK protein expression

Extracellular Signal Regulated Kinase (ERK) protein expression was measured in zebrafish larvae samples using the western blot method and gel doc chemiluminescence. The average results of WB analysis of ERK protein are presented in the form of tables and diagrams as follows (Table 2 and Figure 3).

Table 2. Average Western blot analysis of ERK protein expression.

No.	Treatment	Number of samples (n)	Western blot protein expression of ERK ± SD (μg/μL)	p-value
1.	Normal Group (N)	144	285461.5 ± 22617.5c	<0.001
2.	Insomnia Group (I)	144	211092.5 ± 36867.5b,c
3.	I + 2.5 μg/mL CA	144	94795 ± 30830a
4.	I + 5 μg/mL CA	144	133577 ± 44730a,b
5.	I + 10 μg/mL CA	144	110930.5 ± 40407.5a

Figure 3. Diagram of the average result of WB analysis of ERK protein expression.

Based on the average results of WB analysis of ERK protein expression, the normal and insomnia groups have higher average expression values of 285461.5 ± 22617.5 μg/μL and 211092.5 ± 36867.5 μg/μL compared to different CA extract concentration treatment groups, namely 2.5 μg/mL (94795 ± 30830 μg/μL), 5 μg/mL (133577 ± 44730 μg/μL), and 10 μg/mL (110930.5 ± 40407.5 μg/μL), respectively. Various concentrations of CA extract were able to reduce the average expression value of ERK protein which functions in the mechanism of orexin protein activation as a wake-up trigger in the sleep-wake cycle. Statistical analysis also showed that there was a significant difference in the value of ERK protein expression, this was evidenced by signs of significance in the form of (^a,b,c) which showed that exposure treatment of CA extract was proven to reduce the value of ERK protein expression significantly lower than normal and insomnia groups. The location of the ERK protein is 42 kDa (Table 2 and Figure 3).

Average Western blot analysis of AKT protein expression

AKT Protein expression was measured in zebrafish larvae samples using western blot and gel Doc chemiluminescence methods. The average results of the AKT protein WB analysis are presented in the form of tables and diagrams as follows (Table 3 and Figure 4).

Table 3. Average Western blot analysis of AKT protein expression.

No.	Treatment	Number of samples (n)	Western blot protein expression of AKT ± SD (μg/μL)	p-value
1.	Normal Group (N)	144	181687.5 ± 69844.5a	0.045
2.	Insomnia Group (I)	144	70468.5 ± 34160.5a
3.	I + 2.5 μg/mL CA	144	60113.5 ± 27833.5a
4.	I + 5 μg/mL CA	144	120525 ± 50892a
5.	I + 10 μg/mL CA	144	79935.5 ± 29777.5a

Figure 4. The average of WB analysis of AKT protein expression.

Based on the average results of WB analysis, Akt protein expression can be seen that the normal group and exposure treatment of CA extract concentration of 5 μg/mL have higher average expression values of 181687.5 ± 69844.5 μg/μL and 120525 ± 50892 μg/μL compared to the insomnia group (70468.5 ± 34160.5 μg/μL) and treatment of CA extract concentration of 2.5 μg/mL (60113.5 ± 27833.5 μg/μL) and 10 μg/mL (79935.5 ± 29777.5 μg/μL). The concentration of CA extract 2.5 μg/mL was able to reduce the average Akt protein expression value lower than the insomnia group. Statistical analysis shows that there is a difference in the value of Akt protein expression this is evidenced by the value of p-value = 0.045 (<0.05) which means there is a significant difference, but in post-hoc follow-up tests show that the sign of significance is in the form of (^a) all which means that the average value of Akt expression for each group has no real difference. Therefore, from these results it can be concluded that the treatment of CA extract with the most optimal concentration of 2.5 μg/mL can improve sleep activity in zebrafish larvae insomnia models through decreased Akt protein expression. The location of the Akt protein is 60 kDa (Table 3 and Figure 4).

Average Western blot analysis of p38 protein expression

p38 Protein expression was measured in zebrafish larvae samples using western blot method and gel doc chemiluminescence. The average results of WB protein p38 analysis are presented in the form of tables and diagrams as follows (Table 4 and Figure 5).

Table 4. Average western blot analysis of p38 protein expression.

No.	Treatment	Number of samples (n)	Western blot protein expression of P38 ± SD (μg/μL)	p-value
1.	Normal Group (N)	144	194368.5 ± 20149.5b	0.002
2.	Insomnia Group (I)	144	146856.5 ± 21715.5a
3.	I + 2.5 μg/mL CA	144	123814 ± 9a
4.	I + 5 μg/mL CA	144	117425 ± 6398a
5.	I + 10 μg/mL CA	144	158576 ± 23533a,b

Figure 5. Diagram of the average result of WB analysis of p38 protein expression.

Based on the average results of WB analysis of p38 protein expression, it can be seen that the normal group and the treatment of exposure to CA extract concentration of 10 μg/mL have a higher average expression value of 194368.5 ± 20149.5 μg/μL and 158576 ± 23533 μg/μL compared to the insomnia group (146856.5 ± 21715.5 μg/μL) and the treatment of CA extract concentration of 2.5 μg/mL (123814 ± 9 μg/μL) and 5 μg/mL (117425 ± 6398 μg/μL). The concentration of CA extract of 2.5 μg/mL and 5 μg/mL was able to reduce the average p38 protein expression value lower than the insomnia group. The p38 protein functions in the mechanism of orexin protein activation that can affect the sleep-wake cycle. Statistical analysis shows that there is a significant difference in the expression value of the p38 protein, this is evidenced by the value of p-value = 0.002 or <0.05 which means there is a significant difference. The location of the p38 protein is 38 kDa (Table 4 and Figure 5).

Average total inactivity time (cumulative duration) in zebrafish larvae model of insomnia after exposure to Centella asiatica extract

• a. Light phase

The following are the results of fish motion analysis of total inactivity time (cumulative duration) zebrafish larvae in the normal group, insomnia group (24-hour light exposure), and the group with the administration of CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during the light phase (Table 5 and Figure 6).

Table 5. Average total inactivity time (cumulative duration) of light phase.

No.	Treatment (n=6)	Days-Second (s)			p-value
		5	6	7
1.	Normal Group (N)	933,7497a	720,4394a	99,04633c	<0.001
2.	Insomnia Group (I)	1020,626a	129,0596c	0d
3.	I + 2.5 μg/mL CA	1497,39b	1562,36b	1640,44b
4.	I + 5 μg/mL CA	1673,27b	1723,40b	1725,94b
5.	I + 10 μg/mL CA	1747,18b	1667,41b	1415,90b

Figure 6. Diagram of the average result total inactivity time (cumulative duration) in light phase.

• b. Dark phase

The following are the results of fish motion analysis of total inactivity time (cumulative duration) zebrafish larvae normal group, insomnia group (24-hour light exposure), and group with CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during the dark phase (Table 6 and Figure 7).

Table 6. Average total inactivity time (cumulative duration) of dark phase.

No.	Treatment (n=6)	Days-Second (s)			p-value
		5	6	7
1.	Normal Group (N)	1377,46a	1344,70a	1223,81a	<0.001
2.	Insomnia Group (I)	1891,97b	574,77c	0,00d
3.	I + 2.5 μg/mL CA	1400,61a	1470,24a	1730,97b
4.	I + 5 μg/mL CA	1911,82b	1725,29b	2669,08b
5.	I + 10 μg/mL CA	1747,18b	1667,41b	1415,90a

Figure 7. Diagram of the average result total inactivity time (cumulative duration) in dark phase.

Based on the results of observations of locomotor motion of zebrafish larvae in normal groups, insomnia groups (24-hour light exposure), and groups with CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during light and dark phases, it can be concluded that exposure to CA extract with different concentrations can affect the total inactivity time (cumulative duration) in zebrafish larvae to be longer than the insomnia group (Tables 5, 6 and Figures 6, 7).

Average total latency to first sleep in zebrafish larvae model of insomnia after exposure to Centella asiatica extract

Based on the results of observations of locomotor motion of zebrafish larvae in normal groups, insomnia groups (24-hour light exposure), and groups with CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during light and dark phases, it can be concluded that exposure to CA extract with different concentrations can affect the average total time of first sleep onset (latency to first sleep) in zebrafish larvae to be shorter than the insomnia group. This can be evidence that exposure to CA extract can improve insomnia conditions in zebrafish larvae by shortening the first time of sleep (latency to first sleep) (Tables 7, 8 and Figures 8, 9).

Table 7. Average total latency to first sleep light phase.

No.	Treatment (n=6)	Days-Second (s)			p-value
		5	6	7
1.	Normal Group (N)	8,211105a	1036,7d	1460,799d	<0.001
2.	Insomnia Group (I)	27,3666b	1782,928d	1800d
3.	I + 2.5 μg/mL CA	88,78c	17,93a	40,53c
4.	I + 5 μg/mL CA	0,03a	0,03a	0,04a
5.	I + 10 μg/mL CA	13,60b	20,33a	65,29c

Figure 8. Diagram of the average result total latency to first sleep in light phase.

Table 8. Average total latency to first sleep dark phase.

No.	Treatment (n=6)	Days-Second(s)			p-value
		5	6	7
1.	Normal Group (N)	205,5332c	66,2933b	264,2928c	<0.001
2.	Insomnia Group (I)	24,27997b	319,5865c	1800d
3.	I + 2.5 μg/mL CA	24,37b	0,03a	0,03a
4.	I + 5 μg/mL CA	0,29a	0,03a	0,04a
5.	I + 10 μg/mL CA	0,04a	0,04a	0,04a

Figure 9. Diagram of the average total latency to first sleep in dark phase.

Discussion

Insomnia is primarily characterized by individual dissatisfaction with the duration or quality of sleep accompanied by functional impairment during the day. A person with insomnia usually experiences one or more symptoms such as fatigue, lack of energy, difficulty concentrating, and mood disorders. Insomnia can appear as a major complaint or more often occur along with other medical or psychiatric disorders, such as pain disorders and depression.¹¹ A 30-minute rule is used to meet insomnia criteria, including taking >30 minutes to fall asleep, spending >30 minutes awakening after sleep onset or waking >30 minutes before the desired time and before reaching 6.5 hours of sleep.¹²

The latest concept of insomnia suggests the disintegration of molecules that play a role in alternation of wake and sleep rhythms in the brain. Molecules that play a role in the circadian rhythm of waking up are catecholamines, orexin, and histamine, while those that play a role for sleep, such as γ-aminobutyric acid (GABA), adenosine, serotonin, melatonin, and prostaglandin D2 (PGD2).⁷

Hypocretin (Hcrt) (also known as orexin) is a highly conserved prepro-Hcrt peptide product, with 2 enzymatically cleaved Hcrt peptides: Hcrt 1 or orexin A (33 amino acids) and Hcrt 2 or orexin B (28 amino acids). These peptides are synthesized in a group of neurons in the lateral hypothalamus (Hypo), and their neuronal processes extend widely in the brain. Mammals have 2 Hcrt receptors (HcrtRs) (HcrtR1/orexin A/OA receptor and HcrtR2/orexin B/OB receptor) that are distributed throughout the central nervous system and in peripheral organs. To date, a large amount of evidence suggests that Hcrt is involved in various physiological processes, such as sleep/wakefulness, food intake, and energy homeostasis. The HCRT system has also been reported to regulate reproduction.¹³ Other studies have reported that in zebrafish, as in mammals, orexin signaling is involved in the regulation of many physiological functions, such as sleep/wake cycles, energy homeostasis, and locomotor activity.¹⁴

Hypocretin (Hcrt) or orexin of the nearby hypothalamus synthesize and secrete Hcrts or orexin in the hypothalamus and are recognized primarily as important regulators of sleep or wakefulness, energy homeostasis, and appetite. Other researchers have previously shown that excessive orexin expression can cause insomnia-like phenotypes in zebrafish and that treatments such as orexin inhibition can stimulate feeding behavior in zebrafish. The effects of orexin on zebrafish sleeping and eating activity are very similar to what has been reported in mammals.¹³

The main elements involved in the wake mechanism are orexin or hypocretinergic and histaminergic from the posterior portion of the lateral hypothalamus. Activated orexin will increase phosphorylated ERK1/2 levels. ERK1/2 is rapidly phosphorylated in response to OA and OB, mediated primarily by Gq and slightly via Gi. In addition, dopamine and histamine cell groups have the highest density of hypocretin-2 receptors or OB.¹ Activated H1R (histamine-1 receptor) will stimulate immune responses Th1 and Th2. Histamine H1 receptors are also expressed in skin dendrite cells and keratinocytes in skin tissue, and histamine increases NGF production over human keratinocyte H1R. NGF secretion is due to phosphorylation of protein kinase C, extracelullar signal regulated kinase (ERK), and activation of AP-1 as a result of H1 stimulation.¹⁵

Another study explained that orexin, both OA and OB, also stimulated the activation of Akt kinase in the cortical nerves of hypoxic stress-stressed rats. In addition, Akt kinase also plays a role in several signaling pathways for apoptosis induction and ovarian cancer.³ Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. ERK1/2 and p38 are rapidly phosphorylated in response to OA and OB, mediated primarily by Gq and slightly through the Gi pathway.³

Centella asiatica acts as a neuroprotector against various neurological disorders. The promising potential of Centella asiatica against neurological disorders can be attributed to its antioxidant, anti-inflammatory, anxiolytic, and stress-fighting properties. Centella asiatica in rats created with sleep deprivation for 72 hours significantly improved locomotor activity, anti-anxiety effects, lowered cortisol levels as well as improved neuronal inflammation, and apoptosis response.¹⁶ Centella asiatica provides a calming effect on activity in experimental animals, can increase phenobarbitone which has the effect of inducing sleep time and reducing immobility in experimental animals.¹⁷

Research by Kumar illustrates those 8 days of Centella asiatica treatment provides no benefit for inducing sleep but still shows a protective effect against sleep-induced anxiety (sleep deprivation). The neuroprotective effects of Centella asiatica in sleep deprivation conditions that induce anxiety-like behaviors and the anxiolytic effects of Centella asiatica are modulated by NO (nitric oxide).¹⁶

Another study reported that one of the active compounds of Centella asiatica extract, Asiatic Acid (AA), showed an important role against lipopolysaccharide (LPS)/d-GaIN-induced fulminant hepatic failure with inhibition of oxidative stress and inflammation. The proposed mechanism is related to inhibition in MAPK and NF-КB. This study showed that AA induces anti-inflammatory effects through the activation of antioxidant enzymes by suppressing CCI and MAPK activity (p38, ERK1/2, and JNK, as well as increasing Nrf2 activity).⁴

The effect of one of the active substances of Centella asiatica, namely Asiatic acid (AA) on P13 kinase (P13K)/Akt/mTOR has also been investigated as a therapeutic target in ovarian cancer cells. Phosphorylation of P13K, Akt, and mTOR decreased in AA-treated cells, indicating inactivation of the P13K/Akt/mTOR pathway by AA. Asiatic acid has a target at P13K/Akt/mTOR to prevent growth and induction of apoptosis in ovarian cancer cells.⁵ Another study on the benefits of Centella asiatica against inflammatory reactions is mainly played by Centella asiatica compounds, showed that AA protects human bronchial cells against oxidative and inflammatory damage through mitochondrial stability, decreases ROS and PGE2 production, and suppresses the expression of NADPH oxidase proteins, COX-2, NF-КB p65, and p-p38.⁵

Cumulative duration is the total length of time the fish is asleep or in an inactive condition. The characteristics of zebrafish when asleep are that adult zebrafish stop swimming for 6 seconds, and zebrafish larvae stop for 1 minute, do not move at the bottom or surface, and are less sensitive to external stimuli. Zebrafish larval activity is very minimal at night.¹⁸ Zebrafish have cells that are responsive to light¹⁹ and the duration of exposure to light received by fish leads to a decrease in melatonin production which produces several inputs that promote excessive wakefulness in the brain.¹⁹

Sleep latency or latency to first sleep is defined as the time (both day and night) needed to cause the first sleep.⁹ In this study, it was found that the lengthening of latency to first sleep time or sleep latency in zebrafish larvae models exposed to 24-hour light. Continuous exposure to light can cause this condition, according to the theory that zebrafish have endogenously controlled circadian rhythm behaviors that can be affected by light. In another study obtained with a treatment of 14 hours:10 hours of light: dark, it was found that zebrafish larval activity occurred maximally in the phase of the lights turned on. From this study, the average sleep latency at light ranged from 24.8 to 59.2 minutes on the 6th and 7th day experiments with light exposure during the day.⁹ The regulation of light to the sleep-wake cycle is related to the regulation of melatonin and the hypocretin/orexin system (Hcrtr). As much as 60% of these latency conditions decrease at night, which indicates the formation of hcrtr.¹⁹

Conclusions

Based on this study, it can be concluded that the effect of gotu kola extract (Centella asiatica) in improving the sleep activity of zebrafish larvae (Danio rerio) insomnia model by extending the total inactivity time (cumulative duration) and shortening the duration of the first time of sleep (latency to first sleep) in light and dark phases through inhibition of orexin, ERK, p38 and Akt.