Triple probiotic combination effect on metabolic, oxidative stress, and inflammatory parameters in diabetic population: Systematic review and meta-analysis

Abstract

BACKGROUND

Diabetes is an epidemic condition affecting people globally with a high rate of morbidity and mortality. While various pharmacological and non-pharmacological treatment options are used, diabetes is still a non-treatable condition. Constant attempts are made to identify newer therapies that could support better management of the diabetes condition and improve overall quality of life. Numerous studies have supported the potential role of probiotics therapy in the diabetes condition.

AIM

To evaluate the efficacy of triple probiotic combination on glycaemic, metabolic, oxidative stress, and inflammatory parameters in the diabetic population.

METHODS

Randomized controlled trials evaluating the efficacy of the triple probiotic combination in diabetic patients were identified. The quality of the studies was evaluated using the PEDro scale while biasness between and within the studies was assessed using the Risk of Bias tool. Meta-analysis was conducted using RevMan software, and sensitivity analysis was performed using OpenMeta Analyst software. The study protocol was registered in PROSPERO (CRD42024530999).

RESULTS

Five good-quality clinical trials were included. Low-to-moderate risk of bias was observed. Data from 300 participants were included (150 participants in both groups). Probiotics supplementation significantly improved glycaemic and insulin parameters (glucose level: -23.86 mg/dL; insulin level: -5.02 µIU/mL; HOMA-IR: -1.82 score; QUICKI: +0.02 score; HOMA-B: -16.30 score; P < 0.05 for all parameters), reduced oxidative stress and improved antioxidant parameters [TAC: +92.55 mmol/L; glutathione (GSH): +40.55 μmol/L; nitric oxide (NO): +6.45 μmol/L; malondialdehyde (MDA): -0.48 μmol/L; hs-CRP: -2.24 mg/L; P < 0.05 for all parameters except GSH], and improved lipid parameters (total cholesterol (TC): -3.43 mg/dL; triglycerides (TG): -4.26 mg/dL; low-density lipoprotein: -4.62 mg/dL; very low-density lipoprotein: -0.83 mg/dL; high-density lipoprotein (HDL): +1.83 mg/dL; TC/HDL ratio: -0.25; P < 0.05 for HDL and TC/HDL ratio parameters) compared to the control group.

CONCLUSION

The combination of L. acidophilus, L. casei, and B. bifidum is effective in diabetic patients with cardio/renal complications. Further well-designed studies are warranted to support the results of current research.

Key Words: Diabetes; Probiotics; Glucose; Insulin; Systematic review; Meta-analysis

Core Tip: Diabetes is an endemic condition that has high morbidity and mortality rates. Probiotics are live microorganisms that, when consumed, give healthy benefits to the recipient. The current study evaluated the effect of a triple probiotic combination (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum) in the diabetic population by conducting a systematic review and meta-analysis of randomized controlled clinical trials. The results of the current study suggest that the use of this probiotics combination can provide beneficial effects to patients with diabetes.

INTRODUCTION

The prevalence of chronic disease issues is gradually rising with the rise in the "Western diet and lifestyle." Due to its high prevalence rate worldwide, diabetes is one such endemic metabolic disease condition[1,2]. It is projected that 537 million adults worldwide have diabetes, but by 2045, that figure might rise as high as 783 million[3,4]. Although reduced insulin sensitivity and elevated serum glucose and insulin levels are the hallmarks of diabetes, recent research has also emphasized the critical role that oxidative stress and a low-grade chronic inflammatory state play as risk factors and potential consequences of diabetes[5,6]. These conditions can directly contribute to the development of several chronic metabolic disease conditions, such as obesity[7,8], cardiovascular disease[3], renal and hepatic complications[3,9], ophthalmic defects[3], and neuronal damage as well[3]. Diabetes is a leading cause of mortality, accounting for approximately 11.3% of all fatalities, due to its vast range of complications[3].

Currently, the management of diabetes involves use of a variety of non-pharmacological (such as dietary changes, exercise, and physical activity), as well as pharmacological (such as biguanides, thiazolidinediones, DPP-4 inhibitors, and SGLT-2 inhibitors) treatment options[10,11]. Furthermore, several herbal supplements have shown beneficial effects in lowering insulin resistance and reducing glycaemic load[3]. The European Association for the Study of Diabetes and the American Diabetes Association recommended customized treatment options as the preferred method for the optimal management of diabetes because the symptoms and complications of diabetes vary among individuals, making the "one fit for all" therapeutic approach likely unacceptable[3,12]. Diabetes is still incurable despite all the advances, and managing the illness necessitates a variety of treatments that target the various pathological mechanisms involved[13,14].

The phrase "gut microbiome" refers to the variety of microorganisms in the human digestive system. Although the precise number of these gut microbes is very difficult to measure, some studies estimated that the gut region is home to trillions of microorganisms, which is approximately ten times the number of cells in the human body[15,16]. Microbes can be classified as either harmful or benign (friendly). A condition known as gut dysbiosis is linked to a decrease of beneficial bacteria and an increase of harmful bacteria, which can cause several chronic health issues[15]. There is a bidirectional relationship between the gut microbiome and diabetes that has been the subject of numerous studies. Specifically, changes in the gut microbiome can negatively impact insulin sensitivity and cause diabetes, while increased insulin and serum glucose levels in diabetes can negatively impact the gut microbiome environment and cause gut dysbiosis[17-19]. Therefore, it is conceivable that treatments that have a positive impact on the gut microbiome as a whole may be advantageous in the case of diabetes.

The Food and Agriculture Organization of the United Nations, the Panel of the International Scientific Association for Probiotics and Prebiotics, and the World Health Organization all define probiotics as live microorganisms that, when given in sufficient amounts, offer a range of health benefits to the recipient[15,20]. Due to the vastness of the microbiological world, probiotics today include Lactobacillus, Bifidobacterium, Saccharomyces, Bacillus, Streptococcus, Enterococcus, Pediococcus, Leuconostoc, Escherichia, Lactococcus, Akkermansia, and Faecalibacterium[15,21,22]. Probiotics have been shown to benefit diabetes conditions in several pre-clinical and clinical investigations[23-25]. However, because different probiotics were tested in different dose ranges, it is challenging to generalize the findings of these trials to the actual clinical setting. Based on this idea, the current systematic review and meta-analysis study set out to assess the efficacy of a specific fixed dose triple probiotic combination therapy [containing Bifidobacterium bifidum, Lactobacillus acidophilus, and Lactobacillus casei at a total dose of six billion colony-forming units (CFU) per day] in terms of lipid parameters, insulin, oxidative stress, and glycaemic control in patients with diabetes. This is because several clinical studies have demonstrated the beneficial effects of this combination in treating diabetes, but no prior systematic review and meta-analysis study has brought the findings of these published clinical studies together to assess their efficacy.

MATERIALS AND METHODS

Study conduct

The current study was conducted using systematic review and meta-analysis methodology, adhering to the Cochrane Handbook for Systematic Reviews of Intervention and Cochrane Statistical Method guidelines, while reported as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 checklist[26,27]. The complete study was conducted as per a pre-defined protocol registered in the International Prospective Register of Systematic Reviews (PROSPERO) registry before the study initiation with study ID CRD42024530999. The study questions were framed using the PICOS (population, interventions, comparator, outcomes, study design) framework, as described in Table 1.

Table 1 PICOS criteria for study determination.

Parameter	Description
Population	Adults with diabetes
Intervention	Fixed triple probiotic combination of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium bifidum (either as single therapy or in combination with other therapies)
Comparator	Either placebo, any standard therapy alone, or any other supplementation other than the fixed triple probiotic combination of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium bifidum
Outcomes	1 Glycaemic parameters
	a: Serum glucose level
	b: Serum insulin level
	c: Insulin resistance level (HOMA-IR score)
	d: Insulin sensitivity level (QUICKI score)
	e: Pancreatic β-cell functioning (HOMA-B score)
	2 Antioxidant parameters
	a: Total antioxidant capacity level
	b: Glutathione level
	c: Malondialdehyde level
	d: Nitric oxide level
	e: High sensitivity C-reactive protein level
	3 Lipid parameters
	a: Total cholesterol level
	b: Triglycerides level
	c: Low-density lipoprotein level
	d: Very low-density lipoprotein level
	e: High-density lipoprotein level
	f: Total cholesterol/High-density lipoprotein level ratio
Study design	Randomized controlled clinical study
Study questions	1 Is the triple probiotic combination effective in reducing insulin resistance and improving serum glucose and insulin level in patients with diabetes?
	2 Is the triple probiotic combination effective in reducing oxidative stress and improving antioxidant potential in patients with diabetes?
	3 Is the triple probiotic combination effective in improving lipid parameters in patients with diabetes?

HOMA-IR: Homeostatic model assessment for insulin resistance; HOMA-B: Homeostasis model assessment of β-cell function; QUICKI: Quantitative insulin-sensitivity check index.

Search strategy

Using three online databases (Google Scholar, PubMed, and ScienceDirect), two review authors (VD and JK) independently reviewed the literature from inception till November 2024. With the requirement that the articles be in the English language and be full-text accessible, the search was done to find publications that have discussed the outcomes of pertinent randomized clinical trials. Using a combination of free-text words and Medical Subject Headings terms, a literature search was performed to find research on the effects of triple probiotic supplementation on glycaemic, antioxidant, and lipid parameters in patients with diabetes. The complete search strategy is as follows: ‘((probiotic*) OR (Lactobacillus) OR (Lactobacillus acidophilus) OR (L. acidophilus) OR (L.acidophilus) OR (Lactobacillus casei) OR (L. casei) OR (L.casei) OR (Bifidobacterium) OR (Bifidobacterium bifidum) OR (B. bifidum) OR (B.bifidum)) AND ((glucose level) OR (glucose) OR (insulin level) OR (insulin) OR (insulin resistance) OR (HOMA-IR) OR (insulin sensitivity) OR (QUICKI) OR (beta cell*) OR (B cell*) OR (B-cell*) OR (HOMA-B) OR (total antioxidant capacity) OR (total antioxidant) OR (TAC) OR (glutathione) OR (GSH) OR (malondialdehyde) OR (MDA) OR (nitric oxide) OR (NO) OR (high-sensitivity C-reactive protein) OR (C-reactive protein) OR (C reactive protein) OR (hs-CRP) OR (hsCRP) OR (lipoprotein) OR (low density lipoprotein) OR (LDL) OR (high density lipoprotein) OR (HDL) OR (cholesterol) OR (total cholesterol) OR (TC) OR (triglyceride) OR (TG)) AND ((clinical trial) OR (clinical study) OR (randomized clinical study) OR (randomized clinical trial) OR (RCT))’. The keywords like child, children, infant, neonate, neonatal, animal, mice, rat, hamster, rabbit, dog, review, systematic review, meta-analysis, and narrative review were excluded at each search point using automated software.

Study selection and eligibility evaluation

After an extensive database search and duplicate removal, relevant articles were retrieved. The titles and abstracts of all the retrieved studies were evaluated independently by two review authors (VD and BK) for eligibility. Studies deemed eligible were evaluated using the full text for final inclusion. The complete screening process was as per the defined inclusion and exclusion criteria. Articles that reported the results of randomized controlled clinical trials that evaluated the efficacy of triple probiotic (L. acidophilus, L. casei, and B. bifidum) combination on the glycaemic, antioxidant, and lipid parameters, and available as a full-text article in the English language, were included in the current study. If any article was found to be eligible but the data was unclear, the corresponding author of that particular study was contacted by mail for the relevant study data. The reference list of included studies, potential review articles, and systematic review and meta-analysis studies was additionally searched to identify any grey literature that might have been missed by the literature search process. Articles that presented the results of any in vitro studies, pre-clinical animal model studies, and clinical studies of study design other than randomized controlled design, evaluating the efficacy of different interventions other than the triple probiotic (L. acidophilus, L. casei, and B. bifidum) combination on parameters other than the glycaemic, antioxidant, and lipid parameters, studies with unclear data whose authors did not promptly reply or provides us the complete data of the study, and articles not available as full-text article or available in language other than the English language were excluded from the current study.

Data extraction

Following the study selection process, two review authors (VD and VS) independently performed the data extraction process, and the final data were validated by one review author (JK). Using a pre-designed Excel worksheet, the following study characteristics were extracted from the included studies: Lead author, publication year, study design, participants’ disease indication, and age range (if not provided then it was calculated using the data of age mean and standard deviation), gender, interventions provided, sample size in each group, and duration of the study. Additionally, the data regarding the serum glucose level (in mg/dL), serum insulin level (in µIU/mL), homeostatic model assessment for insulin resistance (HOMA-IR) score, quantitative insulin-sensitivity check index (QUICKI) score, homeostasis model assessment of β-cell function (HOMA-B) score, total antioxidant capacity (TAC) level (in mmol/L), glutathione (GSH) level (in µmol/L), malondialdehyde (MDA) level (in µmol/L), nitric oxide (NO) level (in µmol/L), high-sensitivity C-reactive protein (hsCRP) level (in mg/L), total cholesterol (TC) level (in mg/dL), low-density lipoprotein (LDL) level (in mg/dL), very low-density lipoprotein (VLDL) level (in mg/dL), triglycerides (TG) level (in mg/dL), high-density lipoprotein (HDL) level (in mg/dL), and TC/HDL ratio was extracted in a separate pre-designed excel worksheet.

Study quality and risk of bias assessment

The Physiotherapy Evidence Database tool (PEDro scale) and the Cochrane Risk of Bias (RoB2) tool were used to evaluate the quality of included studies, between-study biases, and within-study biases[3,28]. The PEDro scale evaluates the internal validity, external validity, and statistical sufficiency of the study, based on the following assessment criteria: Eligibility criteria specified, subject randomization, allocation concealment, the similarity of baseline prognosis between groups, blinding of subjects, therapists, and assessors, a primary outcome measurement on ≥ 85% of initial subjects, use of intention-to-treat analysis, use of variability measures and use of between-group comparison methods, following which the studies are categorized to either high-quality study (≥ 8 points), moderate quality study (4–7 points), or low-quality study (≤ 3 points). The RoB2 tool evaluates the study biases on five domains, namely randomization process, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of reported results. Two review authors (VD and BK) independently conducted the PEDro scale evaluation and the RoB2 assessment. The results of the assessments were discussed between the authors, and any discrepancy(s) arose were discussed among the authors by joint consensus.

Statistical analysis

The complete statistical method is described in previous studies[3,28]. In brief, data from eligible studies were evaluated using the RevMan software for conducting meta-analysis and generating forest plots for individual evaluation parameters. The mean difference (MD) was used to identify the overall pooled estimate, and the Higgins’ I²-value was used to identify the heterogeneity among included studies. Heterogeneity was considered significant if I² ≥ 50%, and in such a case, the random effect model was used for the pooled estimate. The leave-one-study-out method was the method of sensitivity analysis to evaluate the effect of individual studies on overall observed heterogeneity and pooled estimate, and was conducted by using the OpenMeta Analyst and RevMan software. Additionally, based on the design and therapy intervention of the included studies, subgroup analysis was performed to evaluate the influence of prebiotics on the observed efficacy of triple probiotic therapy for all evaluated parameters. Publication bias assessment was not conducted due to a limited number of studies included in the final analysis. P < 0.05 was considered to determine significance.

RESULTS

Study selection process, study characteristics and quality assessment

A total of 1044 articles were retrieved after initial literature screening, and after duplicate removal, 988 articles were screened for eligibility. After the complete screening procedure, five studies were included in the current study[29-33]. The study selection process is presented in Figure 1. Data from 300 participants (150 participants in each group) were included in the final analysis. The detailed characteristics of individual studies are presented in Table 2.

Figure 1 PRISMA study selection flowchart.

Table 2 Characteristics of included studies.

Ref.	Population	Age (range in years)	Gender	Sample size (active/control)	Intervention		Study duration
					Active group	Control group
Soleimani et al[29]	Dia-Hem	18–80	Male and female	30/30	Triple probiotic supplement (6 × 109 CFU)	No details present	12 weeks
Soleimani et al[30]	Dia-Hem	18–80	Male and female	30/30	Triple probiotic supplement (6 × 109 CFU) + Inulin 0.8 gm	Starch	12 weeks
Tajabadi-Ebrahimi et al[31]	Dia-CHD	40–85	No details present	30/30	Triple probiotic supplement (6 × 109 CFU) + Inulin 800 mg	Starch	12 weeks
Raygan et al[32]	Dia-CHD	40–85	No details present	30/30	Triple probiotic supplement (6 × 109 CFU)	No details present	12 weeks
Farrokhian et al[33]	Dia-CHD	40–85	Male and female	30/30	Triple probiotic supplement (6 × 109 CFU) + Inulin 800 mg	Starch	12 weeks

CFU: Colony-forming unit; Dia-Hem: Diabetics under hemodialysis; Dia-CHD: Diabetics with coronary heart disease.

The quality of included studies was evaluated using the PEDro scale, with the result presented in Table 3. All of the included studies were good quality studies as all the included studies provided adequate details regarding eligibility criteria, randomization process, allocation concealment, baseline similarity, blinding of subjects and therapists, low dropout rate (< 15%), and statistical sufficiency by performing intention-to-treat analysis, and provided details of point and variable measures data and appropriate statistical analysis to determine difference between the groups. None of the studies provided sufficient details regarding the assessor blinding parameter. The detailed PEDro assessment result, along with the study details and criteria for authors’ judgment, is provided in Supplementary Table 1.

Table 3 Quality assessment of included studies.

Parameters	Ref.
	[29]	[30]	[31]	[32]	[33]
Eligibility criteria	1	1	1	1	1
Random allocation	1	1	1	1	1
Concealed allocation	1	1	1	1	1
Baseline similarity	1	1	1	1	1
Subject blinding	1	1	1	1	1
Therapist blinding	1	1	1	1	1
Assessor blinding	0	0	0	0	0
< 15% dropouts	1	1	1	1	1
Intention-to-treat analysis	1	1	1	1	1
Between-group statistics	1	1	1	1	1
Point measures and variability	1	1	1	1	1
Overall score	10	10	10	10	10

Risk of bias assessment

As presented in Figure 2A, three studies were of low risk of bias[31-33], while two studies were found to have a moderate risk of bias[29,30]. Moderate risk of bias was observed due to deviations from the intended intervention parameters. Considering group evaluation, 60% of studies had a low overall risk of bias, while 40% of studies had a moderate overall risk of bias (Figure 2B).

Figure 2 Risk of bias assessment. A: Risk of bias of within individual studies; B: Risk of bias between studies.

Glycaemic and insulin parameters

The effect of probiotics therapy on serum glucose and insulin levels is presented in Figure 3A and B, respectively. Triple probiotic combination therapy significantly reduced serum glucose level (MD: -23.86 mg/dL; 95%CI: -34.92 to -12.80; P < 0.0001) and serum insulin level (MD: -5.02 µIU/mL; 95%CI: -7.67 to -2.37; P = 0.0002) as compared to control therapy. Significant heterogeneity was observed for serum insulin level (I² = 64%; P = 0.04). The result of sensitivity analysis is provided in Supplementary Table 2. For serum glucose level, none of the included studies had significant effect on overall pooled estimate, while for serum insulin level, the removal of one particular study by Soleimani et al[29] significantly reduced the overall observed heterogeneity (I² value from 64% to 18%) without having significant effect on the pooled estimate. As shown in Table 2, the triple probiotic combination was combined with prebiotic inulin in certain studies. Hence, in order to determine the influence of prebiotic on the efficacy of triple probiotic combination therapy, sub-group analysis was conducted and the results of the same is provided in Supplementary Table 3. The addition of prebiotics did not significantly influenced the efficacy of triple probiotic combination therapy in glucose and insulin level parameters. Considering observed heterogeneity among included studies for insulin level parameter, the probiotics alone therapy showed significant heterogeneity (I² = 87%; P = 0.006), while it was non-significant in probiotics with prebiotics group (I² = 0%; P = 0.45).

Figure 3 Efficacy of triple probiotic combination on glycaemic and insulin parameters. A: Serum glucose level; B: Serum insulin level; C: HOMA-IR score; D: QUICKI score; E: HOMA-B score. HOMA-IR: Homeostatic model assessment for insulin resistance; HOMA-B: Homeostasis model assessment of β-cell function; QUICKI: Quantitative insulin-sensitivity check index.

The efficacy of triple probiotic therapy in improving insulin functioning was evaluated using HOMA-IR and QUICKI scores. Supplementation of triple probiotic therapy significantly reduced insulin resistance (MD: -1.82 HOMA-IR score; 95%CI: -3.29 to -0.35; P = 0.02; Figure 3C) and improved insulin sensitivity (MD: +0.02 QUICKI score; 95%CI: +0.01 to +0.04; P = 0.002; Figure 3D) compared to control group. Significant heterogeneity was observed for both the HOMA-IR and QUICKI parameters. As observed from Supplementary Table 2, sensitivity analysis revealed that the removal of one particular study by Soleimani et al[29] significantly reduced the observed heterogeneity for both the HOMA-IR (I² value from 78% to 24%) and QUICKI (I² value from 85% to 3%) parameters without significantly altering the observed pooled estimate, while the removal of one study by Soleimani et al[30] significantly reduced the pooled estimate to non-significant level (MD: -1.82 to -1.70) for the HOMA-IR parameter. As presented in Supplementary Table 3, the addition of prebiotics did not significantly influenced the triple probiotic therapy effect on HOMA-IR and QUICKI score parameters. Considering observed heterogeneity for HOMA-IR parameter, the triple probiotic alone therapy showed significant heterogeneity (I² = 91%; P = 0.001) while the prebiotics and probiotics combination group showed non-significant heterogeneity (I² = 46%; P = 0.18). Similar results were observed for QUICKI parameter with significant heterogeneity (I² = 94%; P < 0.0001) in probiotics alone group while it was non-significant (I² = 30%; P = 0.23) for the prebiotics and probiotics combination group.

Similarly, the efficacy of triple probiotic therapy on pancreatic β-cell functioning was estimated using the HOMA-B score. As presented in Figure 3E, the triple probiotic therapy significantly improved the pancreatic β-cell functioning (MD: -16.30 HOMA-B score; 95%CI: -25.10 to -7.50; P = 0.0003). The result was found to be homogenous (I² value 0%), while sensitivity analysis was not conducted due to low number of included studies. Sub-group analysis for HOMA-B score was not feasible due to the limited number of studies evaluating effect of probiotics therapy on HOMA-B parameter.

Oxidative stress and inflammation parameters

The serum GSH and TAC level parameters were used to estimate the effect of triple probiotic therapy in improving endogenous antioxidant system activity. Supplementation of triple probiotic therapy significantly improved the serum TAC level (MD: +92.55 mmol/L, 95%CI: +40.87 to +144.22; P = 0.0004; Figure 4A) while it approached significance in case of serum GSH level (MD: +40.55 μmol/L, 95%CI: -3.32 to +84.42; P = 0.07; Figure 4B) compared to control group. Marginally significant heterogeneity was observed for serum GSH level (I² = 53%), while non-significant for serum TAC level. Sensitivity analysis (Supplementary Table 2) revealed that none of the included studies had significant effect on overall pooled estimate for serum TAC level, while the removal of studies by Soleimani et al[30] and Farrokhian et al[33] individually reduced the observed heterogeneity to non-significant level. Similarly, the removal of Farrokhian et al[33] study significantly improved the overall pooled estimate for serum GSH level (MD: +40.55 μmol/L to +59.79 μmol/L; P = 0.07 to 0.005). As per Supplementary Table 3, the addition of prebiotics did not significantly influenced the antioxidant efficacy of triple probiotic therapy for improving TAC and GSH levels. Considering observed heterogeneity, the studies evaluating prebiotics and probiotics combination showed marginally high heterogeneity for TAC level (I² = 55%; P = 0.14), while significant for GSH level (I² = 84%; P = 0.01). No heterogeneity was observed among studies for triple probiotic alone therapy for GSH and TAC parameters.

Figure 4 Efficacy of triple probiotic combination on oxidative stress and antioxidant parameters. A: Serum total antioxidant capacity level; B: Serum glutathione level; C: Serum malondialdehyde level; D: Serum high sensitivity C-reactive protein level; E: Nitric oxide level.

The effect of triple probiotic therapy on serum MDA, hs-CRP, and NO levels is presented in Figure 4C-E, respectively. Compared to control group, the triple probiotic therapy significantly reduced the serum MDA level (MD: -0.48 μmol/L, 95%CI: -0.70 to -0.25; P < 0.0001), serum hs-CRP level (MD: -2.24 mg/L, 95%CI: -3.48 to -1.00; P = 0.0004), while significantly improved the serum NO level (MD: +6.45 μmol/L, 95%CI: +2.09 to +10.81; P = 0.004). Significant heterogeneity was observed for serum hs-CRP level (I² = 59%) and serum NO level (I² = 56%) parameters. As presented in Supplementary Table 2, the removal of Farrokhian et al[33] study increased the heterogeneity to significant level (I² = 27% to 51%) for serum MDA level, while removal of the same study significantly reduced the observed heterogeneity to non-significant level (I² = 56% to 0%) in serum NO level parameters, respectively. In case of serum hs-CRP level, the removal of Raygan et al[32] study significantly reduced the observed heterogeneity to non-significant level (I² = 59% to 0%). None of the included study had significant effect on the overall pooled effect estimate for serum MDA and hs-CRP level parameters, the removal of study by Raygan et al[32] significantly reduced the serum NO level to non-significance (MD: +6.45 μmol/L to 5.62 μmol/L; P = 0.004 to 0.145). The addition of prebiotics to probiotics therapy showed no significant benefits in MDA and NO level parameters, while significant benefit was observed for hs-CRP level parameter. As presented in Supplementary Table 3, the addition of prebiotics to probiotics led to significantly greater reduction in hs-CRP level compared to triple probiotic alone therapy (-3.13 vs -1.28; P = 0.02). Considering heterogeneity among studies, no difference in heterogeneity was observed between sub-groups for the hs-CRP level parameter. Considering MDA level, significant heterogeneity was observed among studies evaluating probiotics alone therapy (I² = 74%; P = 0.05), and for NO level, significant heterogeneity was observed among studies evaluating prebiotics and probiotics combination therapy (I² = 77%; P = 0.04).

Lipid parameters

The efficacy of tripe probiotic therapy in improving lipid parameters in patients with diabetes was evaluated using the serum TC, TG, LDL, VLDL, HDL levels, and the TC/HDL ratio parameters. Although being non-significant, the triple probiotic therapy was capable of reducing the serum TC level (MD: -3.43 mg/dL, 95%CI: -10.46 to +3.61; P = 0.34; Figure 5A), serum TG level (MD: -4.26 mg/dL, 95%CI: -15.26 to +6.74; P = 0.45; Figure 5B), serum LDL level (MD: -4.62 mg/dL, 95%CI: -10.66 to +1.42; P = 0.13; Figure 5C), and serum VLDL level (MD: -0.83 mg/dL, 95%CI: -3.03 to +1.37; P = 0.46; Figure 5D) compared to control group, while significantly improved the serum HDL level (MD: +1.83 mg/dL, 95%CI: +0.29 to +3.36; P = 0.02; Figure 5E) and significantly reduced the TC/HDL ratio (MD: -0.25, 95%CI: -0.45 to -0.04; P = 0.02; Figure 5F) compared to control group. None of the parameters had significant heterogeneity among the included studies, and similarly as presented in Supplementary Table 2, none of the included studies significantly affected the heterogeneity index for the serum TC, TG, LDL, VLDL levels, and TC/HDL ratio parameters, but the removal Soleimani et al[29] and Raygan et al[32] studies individually increased the observed heterogeneity for the serum HDL level parameter to marginally significance level. Similarly, none of the included studies significantly altered the overall pooled estimate for the serum TC, TG, LDL, and VLDL level parameters, while the removal study by Tajabadi-Ebrahimi et al[31] significantly reduced the serum HDL level (MD: +1.83 mg/dL to +1.02 mg/dL; P = 0.02 to 0.265) and significantly increased the TC/HDL ratio (MD: -0.25 to -0.19; P = 0.02 to 0.193), respectively. The results of sub-group analysis of the TC, TG, LDL, LDL, and HDL parameters are presented in Supplementary Table 3, while evaluation of TC/HDL parameter was not feasible due to limited number of studies. While addition of prebiotics to probiotics therapy showed better reduction in the TG and VLDL parameters, the effect was non-significant for all the evaluated lipid parameters. Similarly, no significant effect was observed in observed heterogeneity among studies for the TC, TG, LDL, and VLDL level parameters, while for HDL level, the addition of prebiotics to probiotics combination therapy showed significant heterogeneity (I² = 75%; P = 0.05).

Figure 5 Efficacy of triple probiotic combination on lipid parameters. A: Serum total cholesterol level; B: Serum triglycerides level; C: Serum low-density lipoprotein level; D: Serum very low-density lipoprotein level; E: High-density lipoprotein level; F: Total cholesterol/high-density lipoprotein level ratio.

DISCUSSION

Findings and interpretations

The current study used a systematic review and meta-analysis methodology to assess the effects of a fixed dose triple probiotic combination containing Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum on glycaemic, insulin, lipid, oxidative stress, and inflammation parameters in the diabetic population. The results show that the triple probiotic therapy is successful in lowering insulin and serum glucose levels, perhaps through improvements in insulin sensitivity and decreased insulin resistance. Additionally, the triple probiotic therapy dramatically improved only a few lipid measures while also reducing the parameters associated with oxidative stress and inflammation, potentially through raising endogenous antioxidant levels.

The mechanistic role of probiotics has been evaluated in various studies. Supplementation of probiotics can promote positive effect in diabetes by the following actions: Positively modulate the intestinal environment by increasing the number of beneficial microbes and reducing the number of pathogenic microbes; improve the overall intestinal epithelial junction health and functioning; reducing the level of systemic endotoxin, thereby reduce the deleterious effect on the insulin signalling pathway; improving the production of short-chain fatty acids production and subsequently their level in systemic circulation; improving the level and activity of endogenous insulin secretagogues and insulin sensitizers; reducing the level of carbohydrate absorption; improve intestinal responsiveness to ingested glucose and thereby improving responsive insulin secretion; modulating the intestinal and systemic immune system activity thereby improving the release of anti-inflammatory cytokines and reducing the level of inflammatory cytokines; reducing the oxidative stress and thereby preserve the pancreatic cells health and protect from oxidative stress induced damage[23,34-36]. Due to such myriad of actions, various clinical studies have supported the role of probiotics in diabetic conditions[24,25]. The results of the current study are in line with the results of previous studies demonstrating the positive benefit of the triple probiotic combination therapy in diabetic condition. Prebiotics are ingredients that are consumed by the gut microbes for their pronounced growth and activity. Numerous evidences supports the beneficial role of prebiotics in improving the effectiveness of probiotics therapy[37]. In the current study, the data of certain studies were included that have utilized inulin as a prebiotic along with the triple probiotic combination. Various studies have supported the beneficial effect of inulin therapy in various parameters[38,39]. In order to evaluate the influence of prebiotic inulin on the triple probiotic therapy activity, sub-group analysis was conducted. Based on the observed results, the addition of inulin did not significantly influenced the effectiveness of triple probiotic combination in all the evaluated parameters (except hs-CRP parameter in which the efficacy was significantly improved due to the addition of inulin with the probiotics therapy). This discrepancy observed might be due to numerous reasons. Firstly, the effect of inulin with the probiotics was evaluated for a short time duration. Secondly, previous clinical studies have demonstrated the beneficial effect of inulin therapy at dose of 10 gm/day[40], while the dose of inulin in the current analysis included studies was 800 mg/day. Based on these observations, we hypothesize that the dose and duration of inulin used in the studies included in the current analysis might not be sufficient enough to show additive or synergistic effect along with the triple probiotic regimen, and hence we suggest that more clinical studies can be conducted using higher dose of inulin along with the triple probiotic combination for longer study duration with similar evaluation parameters as that of current study that will provide rational justification to this hypothesis.

Strengths and limitations of current study

The current study has several advantages. The current study is unique since no prior systematic review or meta-analysis has assessed the effectiveness of the specific fixed dose triple probiotic combination (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum six billion CFU daily) in the diabetic population. Numerous previous studies have evaluated the effect of probiotics therapy in diabetic population[41-43], but none of the studies have evaluated the efficacy of this triple probiotic combination therapy on diabetic population. While the result of previous systematic review and meta-analysis studies have supported the use of probiotics in diabetes condition, the generalization of the previous studies’ results to real-world scenario is difficult as the studies have evaluated the effect of different probiotic combinations in diabetes condition. As numerous probiotic combination products are available in market, the selection of appropriate probiotics species and dose in diabetes condition is difficult. The results of the current study are novel and can be used in real-world scenario for better informed decision for the management of diabetes condition. The efficacy evaluation of same probiotics combination therapy in diabetic population helps in better selection of probiotics therapy. Also, the current study's results are consistent with and lend additional credence to the conclusions of earlier research assessing the effectiveness of triple probiotic combination therapy in the diabetic group. The transparency of the current study is also a strength which was made by making the study protocol easily accessible through PROSPERO (CRD42024530999) and publishing the current study in accordance with PRISMA criteria, the study was kept as transparent as feasible. There are also certain limitations to the current investigation. Firstly, there was a notable degree of heterogeneity for some evaluation factors. The leave-one-study-out analysis was conducted to identify the potential cause of observed heterogeneity. The analysis helped us to identify potential studies responsible for the observed heterogeneity, but the exact reason for sub observed heterogeneity among studies was not identified with the provided study data. Additionally, the sub-group analysis was conducted to evaluate whether the observed heterogeneity might be due to the addition of prebiotic inulin along with the probiotics therapy. Even after these methods, the precise cause of heterogeneity is still a matter of further analysis. We believe that additional statistical methods (regression analysis and other) could be performed in future studies that can help identify a more precise cause of observed heterogeneity. Second, none of the included research used assessor blinding (also known as detection bias). The effect of assessor biasness may be minimal because the authors assume that all of the evaluated parameters were laboratory-based evaluation parameters. However, the possibility of potential biasness is raised by the fact that the assessors may be involved in the research study itself. The result of previous studies have demonstrated that non-blinding of assessors can lead to increased pooled effect size by 68%[44,45]. So more rigorous clinical studies with appropriate blinding of the assessors are required to further support the current study’s findings. Third, the current study included data from participants with diabetes along with coronary heart disease or were under hemodialysis. While the parameters evaluated in the current study were related to glycaemic status, insulin resistance, level of inflammatory and oxidative stress, and lipid parameters which are found to be abnormally altered in patients with diabetes without any other co-morbid disease conditions, the results of the current study requires special attention for implementation. Additionally, more clinical studies involving participants with type-1 or type-2 diabetes without any particular co-morbid conditions are required. Also, the number of studies included in the current analysis were limited that prevented the publication bias assessment. Hence, more randomized, double-blind, controlled studies with study design to minimize the potential biasness are warranted to further support the results of the current study. In light to the above discussed strengths and limitations of the study, it can be observed that the results of current study must be interpreted with caution and further clinical studies must be conducted to support the findings of current study.

CONCLUSION

The findings of the current study suggests that the combination of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum might be effective in diabetic patients with associated cardio/renal impairments. However, more carefully planned clinical trials are necessary in light of the previously mentioned study limitations.