The Effects of Probiotic Supplementation on Opioid-Related Disorder in Patients under Methadone Maintenance Treatment Programs

Introduction Patients under methadone maintenance treatment programs (MMTPs) are susceptible to numerous complications (e.g., mental and metabolic disorders). This study evaluated the effects of probiotics on clinical symptoms, biomarkers of oxidative stress, inflammation, insulin resistance, and serum lipid content in patients receiving MMTPs. Materials and Methods A randomized, double-blind, placebo-controlled trial was conducted among 70 patients receiving MMTPs to receive either 1.8 × 109 CFU/day probiotics (n = 35) or placebo (n = 35) for 12 weeks. Clinical symptoms and metabolic profiles were measured before and after the intervention in patients receiving MMTPs. Results Compared with the placebo group, probiotic supplementation resulted in a significant improvement in the severity of depression (P < 0.05). In addition, probiotic administration significantly decreased fasting plasma glucose (FPG), total cholesterol, and low-density lipoprotein cholesterol (LDL cholesterol) (P < 0.05). Furthermore, probiotics resulted in a significant reduction in high-sensitivity C-reactive protein (hs-CRP) and a significant elevation in total antioxidant capacity (TAC) and total glutathione (GSH) levels (P < 0.05). Conclusion Treatment with probiotics for 12 weeks to patients receiving MMTPs had beneficial effects on symptoms of depression, as well as several metabolic profiles. Clinical Trial Registration: this study was registered in the Iranian website (https://www.irct.ir) for clinical trials registration (https://fa.irct.ir/trial/46363/IRCT20170420033551N9). The registration date is March 22, 2020.


Introduction
According to the United Nations [1], more than 180 million people around the world are suffering from drug addiction. One of the most worrying post-addiction problems is malnutrition in all walks of life, whether in industrial or nonindustrial human societies [2]. In addition, overall body mass has been shown to increase predominantly due to an increase in the proportion of body fat upon methadone treatment [3]. A national pilot study was conducted in Iran in 2002 to assess the feasibility of reducing drug-related adverse effects, leading to the establishment of private and public centers for methadone treatment [4]. Several reasons support the efficacy of methadone as an effective treatment for opioid use disorder, including cost-effectiveness and strong potential for managing the physical and psychological condition of opioid-like addiction [5]. Approximately 5,000 outpatient methadone maintenance treatment programs (MMTPs) and buprenorphine exist in Iran to treat opioid dependency, covering nearly half a million subjects [6]. It should be noted that there remain multiple unresolved challenges and questions despite many practical MMTPs. Following the implementation of MMTPs, opioid abusers experienced several metabolic (such as inflammation and oxidative stress) and mental (such as sleep, depression, and anxiety) disorders, which affect their quality of life and increase the frequent drug addiction [7,8]. Treatment with methadone, compared with buprenorphine, was associated with increased frequency of metabolic syndrome. Both drugs are associated with obesity, overweight, and insulin resistance. Extended methadone treatment may cause altered triglycerides, blood pressure, HDL, fasting glucose, and hemoglobin A1C profiles [9]. In addition, previous reports on gut microbiota have explored the effects of drug use on bacterial composition and diversity. Patients under MMTPs altered gut microbiota compared with former and current drug users, likely due to effects of both dietary changes and direct intake of the opiate methadone, leading to enrichment for bacteria (e.g., Bifidobacterium, Fusicatenibacter, and Lactobacillus) and enhanced orexin-A [10].
Gut microbiota represents a diverse number of microorganisms in the digestive tracts, which influence host physiology, including the immune system and the central nervous system [11]. In addition, the gut microbiota is known to be related to opioid use, psychiatric disorders, and type 2 diabetes [12]. e gut microbiota can regulate cerebral performance and behavior by modulating several neurometabolic and neurochemical processes [13,14]. Special attention has recently been paid to probiotics due to wide clinical purposes and health-promoting effects to manage several clinical conditions, such as chronic and acute gastrointestinal and non-gastrointestinal problems [15]. Probiotics have numerous health-promoting effects, and the gut microbiota are vital for the regulation of metabolic and mood processes [16]. Patients with multiple sclerosis showed an improvement in general health, depression, and anxiety and stress scales following twelve weeks of probiotic supplementation in a study by Kouchaki et al. [17]. e results of a meta-analysis in randomized control trials indicated a reduction in depression and anxiety levels in patients supplemented with probiotics [18]. Nevertheless, there are reports on the lack of effect of probiotics on mental health outcomes in obese pregnant women [19]. A systematic review and meta-analysis showed that malondialdehyde level, C-reactive protein, interleukin-10, and Hamilton Depression Scale were positively improved in psychiatric patients supplemented with probiotics [20]. Further, psychiatric patients treated with probiotics exhibited improved levels of HDL cholesterol, VLDL, triglycerides, insulin resistance, insulin, malondialdehyde, and C-reactive protein. [21]. Beneficial activities have been reported for various probiotics, including enhanced absorption of nutrients, overexpression of brain-derived neurotrophic factor (BDNF), decreased levels of pro-inflammatory cytokines, free radical scavenging potential, overproduction of gamma-aminobutyric acid (GABA), regulation of critical neurotransmitters, and antioxidant activities. ese activities may play a pivotal role in the pathology and physiology of metabolic and mental health processes [22][23][24][25][26].
A literature review revealed limited information on the effects of probiotic administration on depression, anxiety, sleep quality, lipid profiles, insulin sensitivity, inflammatory factors, and oxidative stress biomarkers in opioidrelated disorder. Hence, the present research aimed to evaluate the influential activities of probiotics on clinical symptoms, biomarkers of oxidative stress, inflammation, insulin resistance, and serum lipid content among MMTP patients.
(2) Taking probiotic, multivitamin-mineral, and antioxidant supplements during the last 3 months before the intervention.  Pharma Company (Tehran, Iran). Random numbers were generated with computer software (Stat Trek) by trained staff at the Soltan Mirahmad Clinic. Randomization and allocation were concealed from the researchers and patients until the final analyses were completed. A person at the substance abuse treatment clinic, who was not involved in the trial and was unaware of random sequences, assigned the participants to the numbered bottles of supplements. At the clinic, patients received a weekly dose of methadone in the form of syrup, about half of the daily dose in the clinic and the remainder at home.

Assessment of Outcomes.
Mental health is an important indicator of the health status of patients under MMTPs. erefore, mental health (depression, anxiety, and sleep quality) was considered as the primary outcome, and lipid profiles, insulin metabolism, inflammatory factors, and oxidative stress biomarkers were considered as secondary outcomes.

Clinical
Signs. In this study, Beck's Depression Inventory (BDI), Beck's Anxiety Inventory (BAI), and Pittsburgh Sleep Quality Index (PSQI) were used to assess the levels of depression, anxiety, and sleep quality, respectively [27][28][29]. BDI is a 21-question and BAI is a 20-question inventory in which each question was scored between 0 and 4; higher scores indicate higher levels of depression and anxiety. e Persian versions of both inventories were validated in the previous studies [30,31]. PSQI is an applicable instrument used to measure the pattern and quality of sleep. It differentiates poor from good sleep quality by measuring seven components of sleep (e.g., subjective sleep quality, sleep duration, sleep latency, habitual sleep efficiency, use of sleeping medications, sleep disturbances, and daytime dysfunction) over the last month [29].
2.6. Biochemical Assessment. At baseline and after the 12week intervention, fasting blood (10 mL) was collected from each patient at the substance abuse treatment center (Soltan Mirahmad Clinic). Serum insulin levels were measured with an ELISA kit (DiaMetra, Milano, Italy) with intra-and interassay CVs below 5%. e homeostasis model of assessment, QUICKI, and HOMA-IR were assessed using the established formulas [32]. Enzymatic kits (Pars Azmun, Tehran, Iran) with inter-and intra-assay CVs of less than 5% were used to measure fasting plasma glucose and lipid parameters. Serum hs-CRP levels were determined by the commercial ELISA kit (LDN, Nordhorn, Germany) with inter-and intra-assay CVs below 7%. In addition, NO levels were assessed using the Griess method [33]. In addition, TAC levels were assessed using the ferric-reducing antioxidant power method developed by Benzie and Strain [34]. In addition, GSH and MDA levels were evaluated using Beutler et al.'s method and thiobarbituric acid reactive substance spectrophotometric test, respectively [35,36]. CVs for plasma MDA, GSH, and TAC were less than 5%.

Statistical Analysis.
e normality of data was assessed by the Shapiro-Wilk test using the Statistical Package for Social Science Version 22 (SPSS Inc., Chicago, Illinois). To detect the differences in anthropometric parameters between treatment groups, we used the independent-samples t-test and chi-square test. Multiple linear regression models were used to assess treatment effects on study outcomes (clinical sign parameters, lipid profiles, insulin sensitivity, inflammatory factors, and oxidative stress biomarkers) after adjusting for baseline levels of variables. e effect sizes were presented as the mean differences with 95% confidence intervals. P values <0.05 were considered statistically significant.

Results
Of 101 screened patients with MMTPs, 70 patients were enrolled in the study and randomly assigned to either the intervention or control group to receive probiotics or placebo, respectively (35 people to each group). In the placebo group, four patients discontinued intervention due to positive methamphetamine urine test and personal reasons. In the intervention group, seven patients revoked their consents. erefore, 59 patients (intervention (n � 28) and placebo (n � 31)) were analyzed. CONSORT flow diagram on the enrollment of patients in this study is shown in Figure 1. No side effects were ascertained following the administration of probiotics and placebos in patients receiving MMTPs. Patients' characteristics were analogous between the two groups (Table 1).

Clinical Signs.
Compared with the placebo group, probiotics significantly decreased the level of depression (β 1.94; 95% CI, 0.14, 3.75; P � 0.03), respectively. In addition, no significant differences were indicated between the two groups in terms of the Beck's Anxiety Inventory and Pittsburgh Sleep Quality Index (

Discussion
Our study investigated the effects of probiotics on clinical symptoms, lipid profiles, insulin sensitivity, inflammatory factors, and oxidative stress biomarkers in patients receiving MMTPs. e results showed that a 12-week probiotic supplementation in patients receiving MMTPs had beneficial effects on depression, glycemic control, total and LDL cholesterol, hs-CRP serum level, total antioxidants, and plasma glutathione, no effect on anxiety, sleep quality, insulin levels, HOMA-IR, QUICKI, triglycerides, VLDL and HDL cholesterol, malondialdehyde levels, and plasma nitric oxide. Addiction can cause mental health disorder, metabolic disorder, and oral complications and may result in relapse to drug abuse and reduced addiction treatment success rate [7,8,37]. ere seems to be an increasing volume of evidence of an association between metabolic disorder and periodontitis [38]. e periodontal disease represents a progressive destruction of tooth-supporting tissues. Recently, paraprobiotics are regarded as an adjunctive therapy to the nonsurgical scaling and root planning [39]. On the other hand, gut homeostasis plays an important role in human and animal health. e disruption of gut homeostasis enhances the risk of infections and represents potential side effect of opioid use. e effects of opioids in the gut homeostasis, both chronic and acute, include persistent constipation and might worsen pain, mental health signs, and metabolic syndrome [40][41][42]. In addition, the gut microbiota in rats treated with methamphetamine demonstrated that Bacillaceae and Ruminococcaceae were more abundant in the rats with methamphetamine-induced conditioned place preference [43]. So, gut microbial diversity might be shaped by addiction-associated behaviors and these can function as biological indicators to evaluate the health of people with a history of drug use. Also, disturbances in mental health parameters and metabolic profiles have previously been documented in opioid users and patients receiving MMTPs [7,44]. is evidence suggested a potential link involving the gut microbiota, metabolism, and psychological factors that warrant consideration therapeutically. is is the first study to investigate the effects of probiotics on clinical symptoms, lipid profiles, insulin sensitivity, inflammatory factors, and biomarkers of oxidative stress in patients receiving MMTPs.

4.1.
e Effect of Probiotics on Clinical Symptoms. e prevalence of mental disorders in patients receiving MMTPs is ten-fold higher than in the general population and two-to three-fold higher than those with substance abuse. In addition, the high prevalence of sleep disorders in patients receiving MMTPs has been estimated at 84% [45,46]. A growing body of evidence has linked mental health symptoms to the gut microbiome, demonstrating that the latter might be modulating the gut-brain axis [47]. Disturbances in the microbiome have been implicated in both mental and physical illnesses. Signs of psychiatric disturbance, such as depression, anxiety, and autism, have been posited to be associated with disturbed gut microbiota [47,48]. Opioid use significantly affects patients' diets and leads to malnutrition. Numerous studies have reported the effects of drug abuse on nutrition and nutritional deficiencies, and a beneficial effect of probiotics as a food or aromatic plant product in nutrition and diet has been advanced [49][50][51]. e gut microbiome as a therapeutic target for mental health disorder has been a focus in psychiatric research, among many others [52]. In our study, a 12-week probiotic supplementation in patients receiving MMTPs reduced depression scores but had no effect on anxiety scores and sleep quality. It has previously been reported that the use of probiotics has a beneficial effect on mental health and cognitive function [53]. Recent studies in animals and humans have shown that interventions involving intestinal microbiota, including probiotics, may be an effective treatment for mental health disorders [54,55]. In an animal study, Li et al. reported that probiotics may reduce anxiety and depression [56]. Furthermore, young healthy women who received probiotics containing two strains of Lactobacillus helveticus and Bifidobacterium longum for 30 days experienced significant improvements in their mental health [57]. It has also been reported that long-term probiotic supplementation in rugby players may have beneficial effects on their sleep quality and muscle soreness during training  International Journal of Clinical Practice and while playing games [58]. In a meta-analysis of 10 clinical trials, it was shown that probiotics had a marginal effect on mood in general [59]. In addition, no significant improvement was observed in patients with schizophrenia following treatment with Lactobacillus rhamnosus and Bifidobacterium supplements for 14 weeks [60]. ese discrepancies might be due to differences in the strains used or the length of the studies. e exact mechanism of action of probiotics in the brain, and its effects on mental health disorders, has yet to be fully understood. It has been posited that the administration of probiotics may improve psychological symptoms by increasing plasma tryptophan levels, decreasing serotonin metabolite concentration in the frontal cortex, and decreasing dopamine metabolite concentrations in the amygdale [61].

e Effects of Probiotics on Lipid Profiles and Insulin
Metabolism. Impaired insulin metabolism, hypertension, weight gain, and changes in serum lipids in patients receiving MMTPs may lead to long-term side effects, metabolic abnormalities, infections, chronic obstructive pulmonary disease, type 2 diabetes, and higher mortality [62][63][64]. erefore, probiotics may be useful for lipid and blood glucose disorders due to their beneficial effects on lipid and glycemic parameters. Our study showed that consuming probiotics for 12 weeks in patients receiving MMTPs significantly reduced blood sugar, total cholesterol, and LDL cholesterol compared with placebo, but had no significant effect on insulin levels, HOMA-IR, QUICKI, triglycerides, and VLDL and HDL cholesterol. Although the information on the effects of probiotics on lipid and blood glucose profiles in patients receiving MMTPs is limited, several studies have evaluated these profiles in patients not receiving MMTPs. In 2019, Kijmanawat et al. studied GDM and controlled diet in 57 pregnant women. After four weeks of probiotic use, a significant improvement in fasting blood sugar and insulin and insulin resistance levels was observed [65]. In a meta-analysis in 2020, nine studies on the effects of probiotics on neurological diseases were analyzed, showing that probiotics had a significant positive effect on insulin, insulin resistance, triglycerides, and VLDL and HDL cholesterol [21]. In another study in patients with nonalcoholic fatty liver disease, liver enzymes and clinical signs were significantly reduced after 3 months of probiotic use, although no significant changes in triglyceride levels were observed. However, contrary to the results of our study, in this study no significant change in fasting blood sugar was reported [66]. e beneficial effects of probiotics on insulin sensitivity may be due to their effects on the expression of genes associated with fat accumulation and obesity. In addition, the production of short-chain fatty acids (SCFAs) following probiotic use, especially butyrate, stimulated the release of glucose-like peptide (GLP-1) from L intestinal cells and improved glucose tolerance.

e Effects of Probiotics on Inflammatory Biomarkers and
Oxidative Stress. In patients receiving MMTPs, there is a loss of antioxidant capacity and an oxidative imbalance, reflected by increased reactive oxygen species (ROS) generation, matrix metalloproteinase activity, and inflammatory cytokines [67,68]. erefore, probiotics may have beneficial effects on inflammatory profiles and oxidative stress. is study showed that probiotic use for 12 weeks in patients receiving MMTPs significantly decreased serum hs-CRP levels and increased plasma levels of total antioxidants and glutathione compared with the placebo group, but had no effect on plasma nitric oxide and MDA levels. Probiotics had  International Journal of Clinical Practice inhibitory effects on several oxidative stress factors in nervous system disorders in both human [17] and animal models [69][70][71]. Probiotics improved oxidative stress indices, such as SOD and MDA, in an animal model of Alzheimer's disease [70]. In another study in Alzheimer's disease patients, administration for 12 weeks of probiotics containing Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus fermentum reduced MDA, but had no effect on TAC and GSH levels [72].
In a meta-analysis of 12 studies in 2020, Amirani et al. examined the effects of probiotics on metabolic factors in patients with psychiatric disorders, showing beneficial effects on CRP, IL-10, and MDA levels [20]. In addition, 12 weeks of probiotics and vitamin D administration in patients with schizophrenia increased total antioxidants and decreased MDA and hs-CRP [73]. Probiotics decrease oxidative stress via several mechanisms. ese include direct antioxidant capacity, production of antioxidant metabolites, stimulation of host antioxidant activity, increased activity of antioxidant enzyme levels by acting on NF-κB and Nrf2 gene expression, and decreased activity of ROS-producing enzymes such as NADPH oxidase [74][75][76]. In addition, probiotics may affect the expression of cytokines and inflammatory chemokines by acting on TLR receptors and cascading pathways and by acting on genes essential to the inflammatory mechanisms, such as NF-κB [77].

Study Limitations.
Long-term interventions may have better effects on other metabolic profiles, anxiety, and sleep quality. Also, we did not evaluate the dietary intakes of study participants; however, we requested the participants not to change their regular dietary intakes and physical activity. In addition, we did not evaluate craving, pain, urinary profiles, and relapses in patients under MMTPs.

Conclusion
Overall, our novel findings suggest that patients receiving MMTPs and undergoing 12-week probiotic administration experienced improvements in symptoms of depression, but no change in anxiety and sleep quality. In addition, our study showed that probiotics had beneficial effects on blood sugar, total cholesterol, LDL cholesterol, serum hs-CRP levels, total antioxidants, and plasma glutathione, no effect on insulin, HOMA-IR, QUICKI, triglyceride, HDL and VLDL cholesterol, and plasma dialdehyde and nitric oxide levels. Further evidence is needed to demonstrate the efficacy of probiotics in patients receiving MMTPs, better characterizing their mode of action.

FPG:
Fasting plasma glucose GSH: Total glutathione HOMA-IR: Homeostasis model of assessment insulin resistance HDL cholesterol: High-density lipoprotein cholesterol Hs-CRP: High-sensitivity C-reactive protein Data Availability e datasets generated and/or analyzed during this study are not publicly available because the intellectual property is owned by the funding body. ey may be available from the corresponding author on reasonable request containing the approval from the associated funding body.
Ethical Approval e study protocol was approved by the Ethics Committee of Kashan University of Medical Sciences "approval no. IR.KAUMS.MEDNT.REC.1398.116." All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments.

Consent
At the beginning of the questionnaire distribution session, the purpose of the study was explained to the participants, and they were assured about the anonymity and confidentiality of their responses. All participants gave their signed written informed consent letters.

Conflicts of Interest
e authors have no conflicts of interest to declare.

Authors' Contributions
NM, MRA, HM, and AGH contributed in design, conception, and statistical analysis. AHM, HRB, MV, MH, ARS, SAM, PM, and AGH contributed in data collection and manuscript drafting.