Isolation, identification, and characterization of bacterial strains
Despite the isolation of more than 36 bacterial strains, in this research, five bacterial strains which have appropriate probiotic properties were selected. Their physiological and biochemical characteristics were mentioned in supplementary data 1. Based on the standard references and the morphological characteristics of isolated strains, the probiotics were recognized and characterized as Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Bifidobacterium longum and Bacillus coagulans (Supplementary table s1).
Effects of probiotics on biochemical parameters
Biochemical parameters considered for testing probiotics in this study were serum glucose, Ca, P, ALP, BUN, creatinine, and urinary calcium (Fig. 1).
As demonstrated in Fig. 1A, glucose levels significantly reduced after administration of STZ compared to the control group. Blood glucose concentration in the pioglitazone group was significantly lower than the STZ group. Bifidobacterium sp. and Bacillus coagulans significantly decreased the glucose concentrations in rats compared to the STZ group. Bifidobacterium sp. significantly reduced the serum glucose concentrations more than pioglitazone in diabetic rats. No significant changes in serum glucose concentrations were detected for other probiotic strains compared to STZ and pioglitazone groups.
As demonstrated in Fig. 1B, no significant differences in terms of serum calcium concentration between treated groups and control were found.
Similar to calcium, no changes were seen in the serum phosphorus concentration between treated groups and control (Fig. 1C).
Serum alkaline phosphatase (ALP)
Pioglitazone meaningfully declined the serum ALP in comparison with the control group. Interestingly, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus reuteri, Bifidiobacter, and Bacillus coagulans reduced the serum ALP in comparison to the STZ group. Among the groups treated with probiotics, only Bacillus coagulans significantly decreased ALP concentration compared to the pioglitazone group (Fig. 1D).
Blood urea nitrogen (BUN)
In the pioglitazone group, BUN concentration was significantly elevated in comparison with the control group. Again, BUN values were significantly augmented in all groups in comparison with the STZ group, except for the Bacillus coagulans group (Fig. 1E).
The STZ group showed significantly higher serum creatinine levels in comparison with the control group. The high level of serum creatinine was diminished in the pioglitazone, Bifidiobacter, Bacillus coagulans, and Lactobacillus casei groups. However, there was no change in the serum creatinine concentration in the Lactobacillus acidophilus and Lactobacillus reuteri groups compared to the STZ group (Fig. 1F).
STZ, pioglitazone, and the combination of pioglitazone and Lactobacillus acidophilus significantly increased the urinary calcium level in contrast to the control group. Bifidobacteria, Bacillus coagulans, Lactobacillus casei, and Lactobacillus reuteri reduced the urinary calcium concentration to the average level, which was the same as the control group.
Effects of probiotics on DEXA parameters
Besides biochemical parameters, the impacts of probiotic strains on each DEXA outputs (BMD, BMC, and bone area) of global, femur, spine, and tibia were examined.
The probiotics impacts on the global bone area of global, spine, femur, and tibia are revealed in Figure 2. The outcomes exhibited that the global area was significantly reduced in the pioglitazone in contrast to the control group (Figure 2A). Also, probiotics did not significantly improve the global area in the pioglitazone groups in contrast to the control group. In the case of spine-area (Figure 2B), femur-area (Figure 2C), and tibia-BMD (Figure 2D), there were no significant variances between all groups.
The impact of probiotics on the BMC of global, spine, femur, and tibia are displayed in Figure 3. The pioglitazone group and STZ group exhibited significantly low global BMC in contrast to the control group. However, global BMC was notably ameliorated in all probiotics-treated groups in contrast to the pioglitazone group. The global-BMC in the Bifidiobacter group was equal to the control group (Figure 3A).
In the case of spine-BMC (Figure 3B), no substantial variations were detected in STZ, pioglitazone, and probiotics-treated groups compared to the control group. In respect to the femur- BMC (Figure 3C), similar to global BMC, the pioglitazone-treated group and STZ group displayed remarkably low global BMC compared to the control group. However, all the probiotics significantly enhanced the BMC in comparison with the pioglitazone groups and returned it to the normal level. In terms of tibia BMC (Figure 3D), there was a significantly decreased level in the pioglitazone treated group. All probiotic strains enhanced tibia BMC compared to the untreated pioglitazone group, which was only significant in the Bifidiobacter group. No significant changes were observed in terms of tibia BMC after probiotic supplementation, in contrast to the control group; however, probiotics were capable of returning the tibia BMC value to the average level after bone loss induced by pioglitazone.
The impact of probiotics on the BMD of the global, spine, femur, and tibia are reported in Figure 4. Pioglitazone significantly decreased the global-BMD compared to the control group, but all probiotic strains significantly enhanced global-BMD compared to the STZ and pioglitazone groups (Figure 4A). For spine BMD (Figure 4B), a similar trend was observed. No significant variance was spotted between probiotics-treated groups and the control group. Regarding femur BMD (Figure 4C), all probiotics used in this study significantly increased the BMD compared to pioglitazone. In tibia BMD (Figure 4D), despite the significant effect of probiotics on the BMD compared to the pioglitazone group, only Lactobacillus acidophilus and Bifidobacteria sp. revealed BMD as same as control group among the probiotics.