Isolation, identification and characterization of bacterial strains
In spite of 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, Bifidiobacterum 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 were significantly lower than the STZ group. Bifidobacter sp. and Bacillus coagulans significantly decreased the glucose concentrations in rats compared to the STZ group. Bifidobacter 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 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 was significantly augmented in all groups in comparison with the STZ group, except for the Bacillus coagulans group (Fig. 1E).
The STZ group showed significantly greater serum creatinine level 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 combination of pioglitazone and Lactobacillus acidophilus significantly increased the urinary calcium level in contrast the control group. Bifidiobacter, Bacillus coagulans, Lactobacillus casei, and Lactobacillus reuteri improved the urinary calcium concentration to the normal level which was as the same as 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 distinctly examined.
The probiotics impacts on the global bone area of global, spine, femur and tibia are revealed in Figure 2. The outcomes exhibited that global area was significantly reduced in the pioglitazone in contrast to the control group (Figure 2A). In addition, 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 minor 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, pioglitazone-treated group and STZ group displayed remarkably low global BMC compared to the control group, but all the probiotics significantly improved the BMC in comparison with the pioglitazone groups and return it to normal level. In terms of tibia BMC (Figure 3D), there was a significant decreased level in the pioglitazone treated group. All probiotic strains ameliorated tibia BMC compared to the untreated pioglitazone group which was only significant in Bifidiobacter group. No significant changes were observed in terms of tibia BMC after probiotic supplementation in contrast to control group however, probiotics were capable to return the tibia BMC value to the normal level after bone loss induced by pioglitazone.
The impact of probiotics on the BMD of 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 improved global-BMD compared to the STZ and pioglitazone groups (Figure 4A). For spine BMD (Figure 4B), similar trend was observed. No significant variance was spotted between probiotics-treated groups and the control group. In regard to femur BMD (Figure 4C), all probiotics used in this study significantly improved 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 Bifidobacter sp. revealed normal BMD among the probiotics.