Isoflavones and probiotics effect on bone calcium and bone cells in rats

Isoflavones and probiotics have shown the therapeutic potential to alter calcium absorption and bone cell metabolism. This study sought to ascertain the effect of isoflavones and probiotics on calcium status and bone health in healthy female rats. Forty-eight adult female Wistar rats were grouped and fed: a standard diet (control); and standard diets with tempeh; soy; daidzein and genistein; Lactobacillus acidophilus; and a combination of daidzein, genistein, and L. acidophilus. The biochemical serum parameters, such as alanine transaminase, aspartate transaminase, glucose, and triacylglycerol concentrations, were measured, and calcium contents in tissues were determined. After staining the bone with hematoxylin and eosin, the number of osteoblasts, osteocytes, and the percentage of bone marrow adipocytes were counted. Compared with the control group, the soy group showed a significantly lower triacylglycerol concentration. The L. acidophilus group considerably increased the calcium content in the femoral bone. The daidzein and genistein, L. acidophilus, and a combination of daidzein, genistein, and L. acidophilus groups showed significantly lower calcium contents in the heart and kidneys. The daidzein and genistein group significantly enhanced the number of osteoblasts and osteocytes. A substantial inverse correlation was observed between calcium contents in kidneys and osteoblasts. In conclusion, the combination of daidzein, genistein, and L. acidophilus may improve bone calcium concentrations and bone cells. However, no synergistic effect between isoflavones and probiotics was detected in this study.


Introduction
Calcium ions (Ca 2+ ) are vital for living organisms for muscle contraction, pancreatic insulin, and glucagon production in blood glucose (GLU) concentrations [1]. Calcium is also a structurally important component of bones. Therefore, to prevent osteoporosis and fractures by maintaining healthy bones throughout life, a diet rich in calcium and regular weight-bearing physical exercise is necessary [2].  Harahap et al. other modified diets was determined as described in our previous paper [13]. The tempeh (TP) group received AIN 93 M with tempeh flour (250 g/kg of standard diet), whereas the soy group received AIN 93 M with soy Glycine max variety Augusta (RS) flour (250 g/kg of standard diet). The daidzein and genistein (DG) group was provided AIN 93 M supplemented with daidzein (10 mg/kg of standard diet) and genistein (100 mg/kg of standard diet) purchased from LC Laboratories (Woburn, MA, USA). The probiotics (LA) group was provided AIN 93 M supplemented with Lactobacillus acidophilus DSM079 (10 10 CFU/day) obtained from the German Collection of Microorganisms and Cell Cultures, the Leibniz Institute DSMZ, Germany. The daidzein, genistein, and probiotics (DGLA) group received AIN 93 M with daidzein (10 mg/kg of standard diet), genistein (100 mg/kg of standard diet), and L. acidophilus DSM079 (10 10 CFU/day). Tempeh (fermented soy with Rhizopus oligosporus NRRL 2710) and the probiotic (Lactobacillus acidophilus DSM079) used in this study were prepared following the method of Harahap et al. [13]. To standardize the content of daidzein and genistein in this study, the amount of these compounds was adjusted to the content present in 250 g of tempeh. In addition, the proximate nutritional composition of each diet and isoflavones content of soybean and tempeh were reported in our previous result [13].
All groups had free access to diet and distilled water. The rats were weighed weekly, and their food intake was recorded daily. After 8 weeks of intervention, the rats were fasted for 12 h. Then, their body mass was measured, and they were euthanized by decapitation.

Collecting blood and tissues
The rats were treated to a 12-h fast following an 8-week intervention. Following a measurement of their bodily mass, their heads were severed. Immediately upon decapitation, a section was carried out. The total blood sample was collected through cardiac puncture. Dissected tissues were cleaned in saline, weighed, and frozen at − 80 • C. The tissues included the liver, heart, kidney, pancreas, femur, and brain. In addition, hair samples were shaved from the same locations on all rats (the interscapular region). The following biochemical parameters were measured in a commercial laboratory (Alab, Poznań, Poland): alanine transaminase (ALT), aspartate transaminase (AST), glucose (GLU), and triacylglycerol (TG) concentrations.

Determination of calcium contents in diets and organs
Calcium contents in diets were determined following the method of Suliburska et al. [22]. In brief, 2 g of each diet was weighed, ashed in a muffle furnace at 450 • C until complete mineralization, and dissolved in 1 N nitric acid (Suprapure, Merck). Then, calcium concentrations were determined using flame atomic absorption spectrometry (AAS-3, Carl Zeiss, Jena, Germany).
Calcium contents in the liver, heart, kidney, pancreas, femur, brain, and hair were measured after digestion in 65% (w/w) spectra pure HNO3 (Merck, Kenilworth, NJ, USA) using a Microwave Digestion system (Speedwave Xpert, Berghof, Eningen, Germany), following the procedure of Suliburska et al. [23]. After digestion and subsequent dilution with deionized water, calcium contents in the mineral solutions were measured using flame atomic absorption spectrometry (AAS-3, Carl Zeiss, Jena, Germany).
Calcium contents in diets and organs were measured at a wavelength of 422.7 nm. The reliability of this method was confirmed using certified reference materials. A certified reference material of soybean flour INCT-SBF-4 (Institute of Nuclear Chemistry and Technology, Poland) was used to determine calcium contents in diets with a 92% accuracy. In addition, a certified reference material of bovine liver NIST-1577C (Sigma-Aldrich, Saint Louis, MO, USA) was used to determine tissue calcium contents with a 91% accuracy.

Hematoxylin and eosin (H&E) staining bone
Femoral bones used as specimens were fixed in 10% buffered formalin after surgery for 24 h. The samples were then submerged for 3 h in a decalcifying solution (Osteodec bone marrow biopsy decalcifying solution). Then, the bone specimens were treated and embedded in paraffin blocks individually according to the standard operating procedure. Three 2-μm slices were cut from the paraffin blocks and stained with H&E. There were two femoral bone slices with their bone marrow content on each slide. Two researchers independently examined the number of osteoblasts, the number of osteocytes, and the percentage of bone marrow adipocytes of each sample in a high-power field using a light microscope (Leica, Allendale, NJ, USA). Results of ANOVA analysis followed by Tukey's post hoc honestly significant difference test showing significant differences between groups, denoted by different superscript letters ( a, b, c, d, e ) and presented as mean ± SD. *: Calcium concentration in diets was analyzed in triplicate.

Statistical analysis
Tukey's post hoc honestly significant difference test was used to determine statistical significance after conducting an analysis of variance (ANOVA). The Shapiro-Wilk test was used to check the normality of variable distributions. All differences were considered statistically significant at the 5% probability level. SPSS 22 for Windows was used for statistical analysis. The calcium content in diets was measured thrice, and all data were expressed as mean ± standard deviations. Spearman's correlation was used to investigate the association between calcium contents in tissues, blood parameters, and femoral bone cells.

Food consumption characteristics
In all diets, there were no significant differences in the calcium content ( Table 1). The intake of diet was the highest in the daidzein and genistein group and the lowest in the soybean group. Therefore, calcium consumption was also highest in the daidzein and genistein group and the lowest in the soy group. Meanwhile, calcium intake was considerably higher in the daidzein and genistein, L. acidophilus, and the combination of daidzein, genistein, and L. acidophilus groups and significantly lower in the tempeh and soy groups compared with the control group.

Impact on body growth
Body mass and organ mass are presented in Table 2. After the 8-week intervention, the end-line body mass in the groups with modified diets did not differ significantly from that of the control group. The soy group showed a significantly reduced body mass compared with the daidzein and genistein, L. acidophilus, and the combination of daidzein, genistein, and L. acidophilus groups. In contrast to the control and L. acidophilus groups, the soy group showed a significantly higher relative weight of the kidneys. Furthermore, compared with other groups, the soy group showed a significantly higher relative weight of the pancreas. Compared with the tempeh, daidzein and genistein, L. acidophilus, and the combination of daidzein, genistein, and L. acidophilus groups, a significantly higher relative weight of the brain was observed in the soy group.

Impact on selected biochemical parameters
Serum concentrations of biochemical parameters, namely alanine transaminase (ALT), aspartate transaminase (AST), glucose (GLU), and triacylglycerol (TG), are presented in Table 3. Even though there were no discernible differences with the control group, the soy group showed a substantial increase in AST concentrations compared with the L. acidophilus group. The soy group showed significantly lower GLU concentrations than the combination of daidzein, genistein, and L. acidophilus group and significantly lower TG concentrations than the control, daidzein and genistein, and L. acidophilus groups.

Impact on calcium contents in tissues
Calcium contents in tissues after 8 weeks of intervention are presented in Table 4. All treatment groups showed considerably lower Results of ANOVA analysis followed by Tukey's post hoc honestly significant difference test showing significant differences between groups, denoted by different superscript letters ( a, b ) and presented as mean ± SD. *: Relative tissue weights of rats expressed as a percentage of body weight, and relative weight was calculated by dividing tissue weight by body mass and multiplying by 100%.
calcium contents in the heart than the control group. Compared with the control group, the daidzein and genistein, L. acidophilus, and the combination of daidzein, genistein, and L. acidophilus groups showed a noticeable decline in kidney calcium contents. In addition, pancreatic calcium contents were considerably increased in the soy group compared with other groups. The L. acidophilus group showed significantly higher calcium contents in the femur than the control and the combination of daidzein, genistein, and L. acidophilus groups.

Impact on femoral bone cells
Following the 8-week intervention research, the numbers of osteoblasts, osteocytes, and bone marrow adipocytes in the bone femur  Results of ANOVA analysis followed by Tukey's post hoc honestly significant difference test showing significant differences between groups, denoted by different superscript letters ( a, b, c ) and presented as mean ± SD. Results of ANOVA analysis followed by Tukey's post hoc honestly significant difference test showing significant differences between groups, denoted by different superscript letters ( a, b ) and presented as mean ± SD.
of healthy female rats are presented in Table 5, along with the findings of H&E staining (Fig. 2). The numbers of osteoblasts and osteocytes were markedly higher in the daidzein and genistein group compared with the control group.

Correlation between calcium contents in tissues, biochemical parameters in serum, and bone cells
The association between calcium contents in organs, blood parameters in serum, and bone cells is depicted in Table 6. Calcium contents in the kidney showed a significant inverse relationship with triacylglycerol contents and osteoblasts. In addition, pancreatic calcium contents showed a strong inverse relationship with triacylglycerol contents. Meanwhile, glucose and triacylglycerol contents showed a positive relationship with osteoblasts. Moreover, it is important to note that statistical significance (i.e. a low p-value) does not necessarily indicate a strong or clinically significant correlation between variables. In this study, while the p-values for some of the correlations were significant, it is important to consider the strength and direction of the correlation, as well as the context in which it occurs. In some cases, moderate correlations may still have important implications for the research question being investigated.

Discussion
The primary findings of this study were as follows: a modified diet containing daidzein and genistein had significantly high osteoblasts and osteocytes amount, and the presence of L. acidophilus significantly increased the calcium concentration in the femurs of healthy rats.
Numerous studies have reported the effects of daidzein on bone cells and metabolism. For instance, daidzein enhances osteoblast development at various stages (from osteoprogenitors to terminally differentiated osteoblasts), and its influence on bone morphogenetic protein (BMP) production in mature osteoblasts is also reported. BMP2 synthesis is markedly increased in response to daidzein, suggesting that some of the effects of daidzein on the cell may be mediated via increased BMP production by osteoblasts [24]. Similarly, daidzein regulates the growth and development of osteoblastic OCT1 cells by stimulating the BMP-2/Smads pathway [25]. BMP-2 operates on bone cells by binding cell surface receptors and triggering the phosphorylation of Smads, which translocates into the nucleus and then activates the transcription of bone-specific genes [26][27][28]. Furthermore, daidzein promotes bone differentiation, mineralization, and bone formation in both early-and late-stage osteoblasts [29].
Another explanation of these findings may be that daidzein stimulates osteogenesis by facilitating proliferation, differentiation, and antiapoptosis in human osteoblast-like MG-63 cells via the activation of MEK/ERK and PI3K/Akt in an ER-dependent manner [30]. Moreover, isoflavones primarily signal via estrogen receptors, which minimize bone loss by inhibiting osteoclasts [31]. Therefore, the results of the present study are consistent with those of previous studies on the role of isoflavones in enhancing the metabolism of bone cells. Although daidzein and genistein increased osteoblasts and osteocytes, they did not enhance calcium contents in tissues, particularly bone femurs. Bone CYP27B1 gene expression has been shown to be increased in mineralizing osteoblasts and response to a high-calcium diet [32].
In this study, L. acidophilus enhanced the calcium content in the femur in healthy rats. Until recently, information about how probiotic microorganisms potentially influence bone density and mineral metabolism has been scarce [33]. Some probiotics have prevented age-related bone loss in senescence-accelerated mice [34], and others have increased bone mineral density in mice [35]. A decrease in bone mineral density in the femur, the spine, and the whole body has been associated with ovariectomy. Previous studies have shown that L. acidophilus was more successful at preventing this [36]. The present study showed that the calcium content of the femur was significantly higher in the L. acidophilus group compared with the control group. The increased calcium content in the femur and tibia can be attributable to the increased calcium absorption in the distal colon [37]. A clinical study reported that increasing calcium contents in the femur of healthy older women could be attributable to the absence of a dysbiosis effect that could promote bone loss. There is evidence that the gut microbiota regulates bone mass by influencing calcium absorption and the synthesis of serotonin, which interacts with bone cells [38]. The present study's findings suggested that L. acidophilus positively enhances the calcium content in bones, but further studies are needed to confirm these results.
Although daidzein and genistein improved osteoblasts and osteocytes and L. acidophilus increased calcium contents in the femur, in this study, no evidence for a synergistic effect of isoflavones and probiotics on calcium homeostasis and bone formation could be obtained. Perhaps the major reason for this observation is the usage of healthy rats. The condition of bone disorders can reveal synergistic effects. Probably, ovariectomized animal models mimicking osteoporosis might have shown the advantages of isoflavones and probiotics. Previous studies have found that daidzein reduces the osteoclastogenic effect in ovariectomized mice [39] and that L. acidophilus promotes bone heterogeneity and prevents bone loss in osteoporotic mice [14].
In this study, isoflavones and L. acidophilus redistributed calcium between tissues in rats. These findings demonstrate that compared with the control group, the daidzein and genistein, L. acidophilus, and the combination of daidzein, genistein, and L. acidophilus groups showed significantly lower calcium contents in the heart and kidneys (Table 4). However, in these groups, increased calcium contents were observed in the femur. It seems that the combination of daidzein, genistein, and L. acidophilus may regulate calcium redistribution in tissues, especially from the heart and kidneys to bones. Interestingly, this phenomenon can be observed in the inverse correlation between calcium contents in tissues and bone cells (Table 6). A significant inverse association was observed between calcium contents in kidneys and the number of osteoblasts (the lower the calcium contents in kidneys, the higher the number of osteoblasts). The findings of this study suggest that the combination of daidzein, genistein, and L. acidophilus may affect calcium redistribution, which is related to osteoblasts physiology. Moreover, since biochemical parameters regulate the metabolic control system, calcium redistribution affects bone cells associated with blood morphology, in accordance with the positive association observed between blood parameters and bone cells. Evidence from previous studies shows that a high glucose concentration increases osteoblast expression [40]. A significant change was observed in the effect of soybeans on triacylglycerol concentrations. Soybean intake significantly decreased triacylglycerol concentrations compared with the standard diet. This decrease was triggered by the phytate concentration in soybeans [41]. Furthermore, the phytate content of unfermented soy is higher than that of fermented soy products such as tempeh because phytates are mobilized during the fermentation of legumes by Rhizopus during the synthesis of tempeh by the activity of the enzyme phytase, which decreases the concentration of phytic acid [42].
The major contribution of this study is using probiotics and isoflavones together as an intervention to evaluate calcium status in tissues and skeletal architecture of healthy rats. Our findings have important implications for human health as our obtained results suggest that combining isoflavones and probiotics can positively affect bone health by improving bone calcium levels and promoting bone cell proliferation. These findings suggest a potential role for isoflavones and probiotics in improvement of calcium status and in the prevention of osteoporosis, a common and debilitating condition that affects millions of people worldwide. Furthermore, our study provides insight into the potential mechanisms underlying the observed effects of isoflavones and probiotics on bone health, which can aid in the development of more targeted and effective interventions. The results of our research can be the basis for developing nutritional recommendations or dietary supplements for people at high risk of bone diseases due to calcium deficiency.
This study has significant limitations since only selected biochemical and skeletal parameters were analyzed. Moreover, we conducted our study on healthy rats without any bone metabolism disruption. Despite the fact that only healthy female rats were used in this study, we acknowledge the potential limitations of our study concerning gender differences. Our findings provide important insights into the potential benefits of isoflavones and probiotics on bone health. They can serve as a foundation for further investigation in both male and female populations with osteoporosis. Future studies could address the potential gender differences in response to these interventions and further elucidate the underlying mechanisms.

Conclusions
The combination of daidzein, genistein, and L. acidophilus may be potentially beneficial for the calcium content in the bone and bone cells. However, the synergistic effect of isoflavones and probiotics on calcium status and bone health was not observed in healthy rats.

Ethics approval
The Chairman of the Local Ethics Committee for Experimental Animals has permitted the present study.

Consent for publication
All authors consent to the publication of the manuscript.

Availability of data
All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Additional information
No additional information is available for this paper.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.