Oxidative Stress and Gene Expression Dynamics: PENK, PDYN, and NRF2 in Cognitive Recovery from Opioid Withdrawal with Herbal Supplements and Aerobic Exercise

Abstract

Background:

Opioid addiction is a major public health problem that causes impairments in cognitive and oxidative processes. The present study has been designed to investigate the therapeutic potential of aerobic exercise, combined with herbal supplements (Rosa Canina L, Matricaria recutita, and Berberis), on oxidative stress and spatial learning during morphine withdrawal.

Materials and Methods:

This trial consisted of 45 male C57BL/6 mice, divided into nine groups, with the induction of morphine dependence. Herbal supplements were combined with aerobic exercises for a 4-week intervention. Spatial learning was analyzed using the Morris Water Maze test; oxidative stress biomarkers were measured as superoxide dismutase (SOD), malondialdehyde (MDA), and total oxidative status (TOS); and gene expression levels of proenkephalin (PENK), prodynorphin (PDYN), and nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated using real-time polymerase chain reaction (PCR).

Results:

Morphine-dependent mice exhibited impaired spatial learning and elevated oxidative stress. Herbal supplements and aerobic exercise, separately, enhanced spatial learning and antioxidant capacity, but the most significant benefits were seen when combined in reducing escape latency and improving SOD and TOS levels. Gene expression analysis revealed that the combination treatment brought PENK, PDYN, and NRF2 levels closer to those of the control group, suggesting synergistic neuroprotective effects.

Conclusions:

The combination of herbal supplements and aerobic exercise is promising in reducing oxidative stress and improving cognitive recovery during morphine withdrawal. These findings point to a holistic approach in addressing opioid addiction and may inform future clinical applications in improving rehabilitation outcomes and quality of life among affected individuals. Further research is needed to optimize these interventions for broader application.

INTRODUCTION

Opioid addiction is a complex disorder involving psychological and physiological dysfunctions, primarily caused by prolonged use of psychoactive substances. Individuals often transition from seeking the pleasurable effects of opioids to managing distressing withdrawal symptoms.[1] The healthcare system faces immense challenges due to the widespread prevalence of opioid addiction, such as morphine dependence, leading to significant social and economic burdens.[2] Given the rising use of opioids and the persistent relapses associated with addiction, effective treatment strategies are urgently needed to address the multifaceted impacts of opioid dependence[3,4]

Opioid addiction treatments include medication-assisted therapies (MATs), behavioral interventions, and relapse prevention.[5] Methadone, a common MAT, alleviates withdrawal symptoms but can cause mental health issues and other side effects with prolonged use.[6,7] Growing interest in complementary therapies, like herbal remedies, offers potential for reducing withdrawal symptoms and oxidative stress with fewer side effects.[8]

Herbal supplements like Rosa Canina L, Matricaria recutita, and Berberis help control opioid addiction. Rosa Canina L reduces oxidative stress and anxiety, while Matricaria recutita decreases symptoms of withdrawal through interaction with the gamma-aminobutyric acid (GABA) receptor.[9] Berberis presents anti-inflammatory, antioxidative, and antianxiety properties; hence, Berberis is effective during opioid withdrawal. These can be used as promising complementary therapies.[10]

Aerobic exercise plays a vital role in cognitive recovery and reducing oxidative stress by enhancing dopamine and endorphin release, improving mood, and promoting neurogenesis.[11] It reduces drug tolerance and dependency, proving effective in addiction management.[12] Combining exercise with herbal remedies enhances therapeutic effects on cognitive function and oxidative stress, yet their joint impact on morphine withdrawal needs further exploration.[13]

The Morris Water Maze (MWM) is commonly accepted to be a valid behavioral measure for the assessment of spatial learning and memory in rodents. It has been used extensively in the study of cognitive deficits associated with various neurological disorders and addiction. For example, significant increases in escape latency and decreases in path efficiency have been reported in morphine-dependent animals, indicating that changes in hippocampal functioning have occurred due to addiction and withdrawal processes.[14,15]

Similarly, biomarkers of oxidative stress, such as superoxide dismutase (SOD), malondialdehyde (MDA), and total oxidative status (TOS), become vital tools for the estimation of cellular impairment and antioxidant defense functionality. Of note, SOD is one of the key protective mechanisms against reactive oxygen species; its activity has previously been shown to significantly decrease in opioid dependence models, thus showing an increase in oxidative stress.[16] Moreover, interventions augmenting SOD level have been associated with better cognitive and neuronal recovery, underlying its usefulness in addiction and withdrawal researches.[17]

Proenkephalin (PENK) and prodynorphin (PDYN) are considered key genes of the endogenous opioid system, which modulates pain, reward, and stress responses. Nuclear factor erythroid 2-related factor 2 (NRF2) acts as a master regulator of the antioxidant defense system. Opioid dependence compromises PENK and PDYN gene expression, thus leading to cognitive impairments associated with maladaptive neuroplasticity, and reduces the activity of NRF2, which heightens oxidative stress and neuronal damage. Analysis of the expression levels of these genes gives important information on the recovery of the opioid system, restoration of antioxidant defenses, and global cognitive and oxidative recovery during withdrawal, further highlighting the therapeutic potential of the combination therapies. This study examined the interaction between aerobic exercise and herbal extracts in morphine dependence in mice.

MATERIALS AND METHODS

Aim, design, and setting of the study

The combined effects of aerobic exercise and herbal supplementation on oxidative stress, spatial learning, and the gene expression of morphine-dependent mice were investigated. The study was set up as a randomized controlled post-test study. It included a control group to help compare the results. This was done to assess how the interventions affected spatial learning, oxidative stress markers, and gene expression.

Characteristics of samples

In this study, 45 male C57BL/6 mice, aged 6 weeks and weighing 25–30 g, were used. The animals were obtained from the Royan Institution in Isfahan. The mice were kept in Plexiglas cages under controlled conditions of temperature (20–23°C), humidity (35–55%), and a 12-hour light-dark cycle with ad libitum access to food and water.

Processes, interventions, and comparisons

This study investigated the interactive role of aerobic exercise and herbal extracts (Rosa Canina L, Matricaria recutita, Berberis) on morphine dependence in mice. In a randomized controlled design, 45 male C57BL/6 mice were assessed for 4 weeks on spatial learning using the MWM, biomarkers of oxidative stress, namely, SOD, MDA, and TOS, and gene expressions PENK, PDYN, and NRF2. Table 1 lists the 9 groups used in this study.

Table 1.

Nine groups used for tests

Group No.		Description
1		Control
2		Morphine-addicted (MA)
3		Opioid withdrawal using methanol
4		Opioid withdrawal using herbal supplement
5		Opioid withdrawal using aerobic exercise
6		Opioid withdrawal using herbal supplement and aerobic exercise
7		Opioid withdrawal using methanol and herbal supplement
8		Opioid withdrawal using methanol and aerobic exercise
9		Opioid withdrawal using methanol, herbal supplement, and aerobic exercise

Behavioral and molecular assessments

Spatial learning was evaluated by the MWM test, in which mice had to locate a submerged platform over 5 consecutive days; the escape latency was recorded as a measure of learning ability. The biomarkers of oxidative stress measured were SOD, MDA, and TOS. The expression levels of the genes PENK, PDYN, and NRF2 were analyzed in hippocampal tissue samples using quantitative real-time PCR (qPCR) with the SYBR Green method. Extraction of RNA was done by the TRIZOL reagent and cDNA synthesis using the Fermentas kit protocols.

Statistical analysis

Statistical analysis was performed using one-way ANOVA to compare group differences for spatial learning and oxidative stress biomarkers, SOD, MDA, and TOS, and gene expression levels, PENK, PDYN, and NRF2. Post hoc comparisons were carried out with Tukey’s test to evaluate significant differences among groups. The level of statistical significance was considered at P < 0.05.

RESULTS

Table 2 summarizes the behavioral and oxidative stress data across the groups. In the MWM, the control group had optimal spatial learning, represented by the shortest escape latency (36 ± 3.2 seconds), whereas morphine-addicted mice demonstrated severe cognitive impairments, as reflected in the latency of 112 ± 10 seconds. Combined herbal supplements and aerobic exercise reduced the latency time to 62 ± 2.6 seconds, suggesting a synergistic effect [Figure 1]. The SOD level was the highest in the control group at 18.24 ± 1.7 (U/mg protein). Compared to the controls, there was a marked reduction of SOD protein levels in the morphine-addicted group at P < 0.001. The combination groups restored their SOD levels and were significantly higher than the morphine-addicted group at P < 0.01. On the other hand, MDA levels were high in the morphine-addicted group at P < 0.001, while MDA levels were reduced in the treated groups with P < 0.01. The TOS showed noteworthy enhancements with the application of combined treatments (P < 0.05).

Table 2.

Descriptive statistics of behavioral tests and oxidative stress biomarkers across different treatment groups

Group		MWM Mean±SD		TOS Mean±SD		MDA Mean±SD		SOD Mean±SD
Control		36±3.2		3.58±0.87		0.52±0.09		18.24±1.7
Morphine-addicted		112±10		14.49±0.34		2.82±0.07		6.3±0.65
Methadone		91±6		15.52±0.62		3.83±0.065		4.17±0.26
Herbal supplements		98±4.6		12.6±0.85		1.96±0.02		9.52±0.56
Aerobic exercise		77±5.2		12.94±0.38		1.93±0.06		9.29±0.45
Herbal supplements + Aerobic exercise		62±2.6		9.98±0.98		1.23±0.09		11.38±0.89
Methadone + Aerobic exercise		51±1.9		7.2±0.54		0.75±0.056		13.06±0.96
Methadone + Herbal supplements		49±2.3		7.5±0.65		0.71±0.08		13.01±0.52
Methadone + Herbal + Aerobic exercise		39±0.6		5.98±0.23		0.46±0.03		16.41±0.56

Figure 1.

MWM test

The latency to escape during the MWM test was significantly increased in the morphine-addicted group compared with the control group (P < 0.001). Treatments with herbal supplements and aerobic exercise showed separate improvements in spatial learning compared with the morphine-addicted group (P < 0.01), with the highest improvement observed in the combined treatment group (P < 0.001) [see Figure 2].

Figure 2.

Performance in the MWM test across different treatment groups

Gene expression analysis focused on PENK, PDYN, and NRF2, key markers of addiction and antioxidant defense [see Table 3]. PENK expression was significantly elevated in the morphine-addicted group compared to controls (P < 0.001). Combination treatments progressively decreased PENK expression, with the lowest levels observed in the methadone + herbal supplement + aerobic exercise group (P < 0.01). Similarly, PDYN expression was significantly reduced in the morphine-addicted group (P < 0.001) and improved with treatment (P < 0.05). Nrf2 expression, essential for antioxidant defense, was significantly lower in the morphine-addicted group compared to controls (P < 0.001) but was restored to near-control levels in the combination treatment group (P < 0.01) [Figure 3].

Table 3.

Relative expression of genes across different treatment groups

Group		PENK Mean±SD		PDYN Mean±SD		NRF2 Mean±SD
Control		1±0.16		1±0.052		0.97±0.023
Morphine-addicted		8.9±0.35		0.13±0.02		0.12±0.003
Methadone		7.1±0.15		0.29±0.032		0.05±0.006
Herbal supplements		5.36±0.47		0.44±0.012		0.23±0.02
Aerobic exercise		5.21±0.12		0.45±0.03		0.25±0.01
Herbal supplements + Aerobic exercise		4.12±0.18		0.59±0.015		0.36±0.06
Methadone + Aerobic exercise		3.35±0.36		0.73±0.015		0.45±0.06
Methadone + Herbal supplements		3.1±0.18		0.75±0.019		0.48±0.025
Methadone + Herbal + Aerobic exercise		2.14±0.45		0.98±0.016		0.65±0.01

Figure 3.

Relative expression of (a) PENK, (b) PDYN, and (c) NRF2 for different groups

While significantly downregulated in the morphine-addicted group, treatments improved PDYN and NRF2 expressions, reaching near-control levels in the combination group (PDYN: 0.98 ± 0.016, NRF2: 0.65 ± 0.01) [Figure 3]. These improvements were supported by oxidative stress biomarkers [Figure 4], with the morphine-addicted group showing the lowest SOD level (6.3 ± 0.65) and the highest MDA (2.82 ± 0.07) and TOS (14.49 ± 0.34) levels, indicating severe oxidative damage. Combined treatments effectively enhanced antioxidant defenses and mitigated damage. These improvements were supported by ox [Figure 4].

Figure 4.

Concentration of (a) SOD, (b) MDA, and (c) TOS for different groups

While the individual treatments improved the parameters of oxidative stress, the combination of aerobic exercises and herbal supplements caused the most pronounced effects: The SOD level significantly increased in the combined treatment group to 16.41 ± 0.56, approaching the control group levels of 18.24 ± 1.7. At the same time, MDA decreased to 0.46 ± 0.03 and TOS was reduced to 5.98 ± 0.23 in the combined treatment group, suggesting substantial mitigation of oxidative stress.

DISCUSSION

In this study, the effects of aerobic exercise and herbal supplements on spatial learning, oxidative stress, and gene expression were investigated in morphine-addicted mice. The MWM test showed severe impairments in spatial learning and memory in the morphine-dependent group, as revealed by prolonged escape latencies compared to the control group (P < 0.001). Such deterioration in performance might indicate impairments in cognitive functions, which are most likely associated with hippocampal dysfunction due to oxidative stress and neuroadaptations in response to opioid exposure. Both aerobic physical exercise and herbal dietary supplements alone improved spatial learning (P < 0.01), while the combined treatment given together resulted in the greatest recovery (P < 0.001). Our findings are supported by previous studies, which report a promoting effect of aerobic exercise on cognitive function through increased hippocampal neurogenesis and enhanced synaptic plasticity. Botanical supplements also facilitate cognitive recovery by reducing inflammation and oxidative stress, which play critical roles in the recovery of hippocampal function.[18,19]

Oxidative stress markers like SOD, MDA, and TOS were significantly altered in the morphine addiction group, indicating severe oxidative damage. SOD levels were dramatically reduced (P < 0.001), whereas MDA and TOS levels were significantly increased (P < 0.001), reflecting powerful lipid peroxidation and generalized oxidative imbalance. This agrees with evidence suggesting that opioid addiction may exacerbate oxidative stress, possibly resulting in neurodegeneration and cognitive impairment.[20,21]

Combination treatments involving aerobic exercise, herbal supplements, and methadone significantly restored SOD levels and reduced MDA and TOS, approaching control values (P < 0.01). The synergistic effects can be attributed to the complementary mechanisms of action. Aerobic exercise enhances endogenous antioxidant defenses through the activation of pathways like NRF2, which regulates antioxidant response genes.[22] Concurrently, herbal supplements provide exogenous antioxidant compounds, including flavonoids and phenolic acids, which act to neutralize reactive oxygen species.[23]

Gene expression analysis further elucidated the molecular mechanisms underpinning the observed improvements. PENK and PDYN, key components of the endogenous opioid system, were dysregulated in the morphine-addicted group, with significantly elevated PENK levels (P < 0.001) and reduced PDYN levels (P < 0.001). These disruptions are indicative of opioid-induced neuroadaptive changes that impair stress regulation and reward processing.[24,25] These gene expressions were gradually normalized by the combination treatments, indicating recovery of the endogenous opioid system (P < 0.01).[26,27] NRF2 expression, essential for the maintenance of redox homeostasis, was also markedly decreased in the morphine-addicted group (P < 0.001), but its level was significantly normalized with all the interventions (best with combination treatment; P < 0.01).[28]

The superior efficacy of combining aerobic exercise and herbal supplements with methadone can most likely be attributed to their complementary mechanisms. Although methadone is potent in reducing withdrawal symptoms, it does not affect oxidative stress and cognitive deficits. Adding aerobic exercise enhances BDNF levels, leading to neurogenesis and improved cognition.[17] Herbal supplements like Rosa Canina L., with its high content of vitamin C and flavonoids, acts directly to oppose oxidative damage, while Matricaria recutita modulates the GABAergic system, which helps reduce anxiety and withdrawal symptoms.[29]

CONCLUSION

This study showed novel findings where this combination of aerobic exercise and antioxidant-rich herbal supplement integrated with methadone treatment produced superior recovery outcomes in morphine withdrawal. More importantly, synergism significantly alleviated oxidative stress—showing improved levels of SOD and decreased levels of MDA and TOS—and enhanced recovery of cognition with improved performance at the MWM test. Furthermore, combination therapy restored the expression of genes that are crucial for the maintenance of oxidative homeostasis and the endogenous opioid system, including PENK, PDYN, and NRF2. The findings give opportunity to the integration of complementary therapies with standard opioid withdrawal treatments, in order to tackle the complex difficulties associated with addiction recovery. The innovative combination of aerobic exercise and herbal supplements alongside methadone offers a holistic approach that might increase the effectiveness of therapy and improve patients’ outcomes in clinical settings. More research is needed to optimize these interventions for broader implementation.

Ethics approval and consent to participate

All procedures involving animal subjects were performed in compliance with ethical guidelines. The study received approval from the Animal Research Committee of Islamic Azad University, Khorasgan Branch (IR.IAU.KHUISF.REC.1401.109).

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Authors’ contributions

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by Miss Zibandehpour, and Dr. Taghian. The first draft of the manuscript was written by Miss Zibandehpour and Dr. Dehkordi.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.