The ameliorative role of specific probiotic combinations on bone loss in the ovariectomized rat model

Osteoporosis, a skeletal disease described by impaired bone strength that causes an increased risk of fractures, is a worldwide disease that mainly occurs in postmenopausal women and older adults [1, 2]. The condition is a bothering factor for about 200 million people, with low bone mass, worsening of the fine structure of bone tissue, and elevated chance of fractures [3]. Since the population is getting old, it is predictable that the prevalence of osteoporosis will significantly increase [4].

Probiotics are living microorganisms that bring health benefits to consumers by promoting intestinal balance [5, 6]. They contain different microbes explained by their genus, species, and strain designation [7]. Probiotic supplements are beneficial for humans and animals and can be used after antibiotic therapy to compensate for gut microbiome deficiency or as prophylaxis [8]. The International Scientific Association of Probiotics and Prebiotics (ISAPP) has a helpful definition of probiotics consisting of three properties; probiotics should be alive during administration, be healthy for users, and have the right dose when delivered [9].

Many experiments have been recently done to specify the potential profits of probiotic preparations in pathologic bone disorders such as osteoporosis. In this regard, several probiotic strains have been introduced in a single administration or as food supplementation to ameliorate bone loss [2]. Oral intake of a single probiotic strain Limosilactobacillus reuteri (L. reuteri) for 28 days by male rat caused substantial health benefits for trabecular bone density, number, thickness, and bone mineral content and density both in the femur and vertebral [10]. A study showed that when a single strain of Bifidobacterium longum (B. Longum) was orally administered to male rats for 4 weeks, the microelements essential for bone health, such as calcium, phosphorus, and magnesium, were concentrated in the tibia [11]. Also, Lacticaseibacillus paracasei (L. paracasei) could increase cortical bone mineral content, and the resorption marker C-terminal telopeptides in rat blood were decreased as well as calcium exertion through urine [12]. This probiotic strain stimulates differentiation of osteoblasts via bone morphogenetic protein (BMP) and inhibits RANKL-induced differentiation of osteoclast, thus helping to inhibit bone loss. Dar et al. reported that Lactobacillus acidophilus (L. acidophilus) improves the microarchitecture of both trabecular bone and cortical bone, enhancing bone mineral density and heterogeneity immunomodulatory impact on the host immune system [13]. Also, we observed that the supernatant of Bacillus coagulans (B. coagulans) enriched the tibia bone mineral density (BMD) of OVX rat in the previous study [2]. In addition, our previous studies evaluated the effects of isolated several native strains of probiotics on bone loss in ovariectomized rats [3]. Although their mechanism of action hasn’t been entirely understood, these probiotics can affect the regulation of luminal pH, production of enzymes, organic acids, and antimicrobial peptides, improvement of barrier function by enriching mucus secretion, stimulation of osteoblast differentiation, and maintaining of the host immune system [2, 14]. They also may have effects on preventing and treating osteoporosis by affecting calcium absorption via decreasing pH, inhibiting calcium binding to bile acid and elevating the surface area for absorption in the large intestine, regulating the immune responses, and producing small molecules, for instance, serotonin or estrogen-like molecules [15‐17].

Combining several probiotic strains appears to have greater efficacy against many diseases than single-strain therapy. Few studies have shown that multi-strain probiotics, including the strains that are a component of the combination itself, are more potent [18]. However, it is still unclear whether this superiority is because of synergistic effects between the strains or because of the high dose of probiotics used.

To accurately demonstrate the greater efficacy of multivariate probiotics and which multivariate probiotic compound is more effective for treating osteoporosis, further studies using the same doses in similar populations are needed. Given the positive effect of probiotics alone or in randomized combination in the treatment of osteoporosis, we aimed in this study to clarify which particular wise combination of probiotics has the most beneficial effect in the postmenopausal rat model. Therefore, due to the positive impact of probiotics alone in the treatment of osteoporosis, we configured them in seven selective combinations and compared their effectiveness in protecting rats from ovariectomized (OVX)-induced bone loss. As there are several probiotic combinations in the supplements market, this study aimed to determine which probiotic combination is more effective in ameliorating bone loss in OVX rats using quantitative indicators.

Bone is a pivotal system of the human body, and its hemostasis is related to intestinal flora because it is reported that the gut microbiome can considerably affect bone physiology [19, 20]. Maintaining the gut flora equilibrium is achieved by dietary changes or using probiotics and their metabolites (oligosaccharides, carbohydrates, fibers). Probiotics can change the gut microbiota composition, induce anti-inflammatory responses, endorse intestinal calcium absorption, and thus increase BMD [20].

Previous research has unequivocally shown that the intestinal system significantly impacts bone health. One way this happens is by controlling the absorption of minerals like calcium and phosphorus, which are essential for strong bones. Additionally, incretins and serotonin, which are obtained from the gut, and endocrine variables that affect the absorption of these minerals, might affect bone turnover [21, 22]. More recent research has shown how the intestinal microbiome affects bone function using germ-free rat and probiotics [23]. Rats treated with yogurt containing L. casei, L. reuteri, and L. gasseri enhanced calcium absorption, resulting in raised BMC relative to the control in research similar to the mouse model [24]. Similarly, supplementing developing rats with L. rhamnosus (HN001) enhanced calcium retention [25]. B. longum has been found to affect bone in addition to several Lactobacillus strains positively. Male rats supplemented with B. longum (ATCC 15,707) for 28 days had a higher percentage of fracture strength and more calcium and vitamin D in their tibias than untreated rats did [11]. In a different study, rats with a high-cholesterol diet plus fermented broccoli for 12 weeks showed a decrease in the number of TRAP-positive osteoclasts compared to untreated rats [26]. The probiotic combinations used in this study (groups 4, 6, and 7) that included L. acidophilus, B. longum, L. reuteri, L. casei, and B. coagulans significantly enhanced Vitamin D and Ca absorption.

The use of probiotic compounds is believed to have several potential advantages over single-strain formulations, including a greater chance of successful treatment by increasing probiotic strains, more potential niche, and a more comprehensive range of efficacy due to the greater diversity of strains, additive or synergistic effects due to increased adhesion, creation of a favorable environment and reduce intestinal microbiota antagonism [27]. However, the probiotic combination is not always successful and can sometimes have a potentially harmful effect due to the antagonistic effects between the probiotics in the product. Therefore, designing an experiment using multidimensional settings of multi-strain probiotics for such studies can be very useful [27]. Some preliminary studies reported that a VSL#3 (containing three species of Bifidobacterium (B. longum, B. breve, and B. infantis), four strains of Lactobacillus species (L. casei, L. plantarum, L. acidophilus, and L. delbruekii subsp. bulgaricus) has been used to treat osteoporosis [28, 29]. These in-vivo studies showed that this branded combination obviously improved the femoral bone density, trabecular thickness, and number [30]. This may be due to the spinal bone volume after ovariectomy in rats treated with VSL#3.

Although numerous investigations have studied the effect of a probiotic combination in the prevention and treatment of osteoporosis in animal models, there is much diversity among their strains. Since most of these compound strains are naturally present and formulated, there is no consensus on the most effective probiotic compound. Among the studies that examined the effect of probiotic combinations on the prevention and treatment of osteoporosis, we discussed in depth the familiar strains. Despite all the differences and similarities, our research team finally concluded that common strains found in the most effective probiotic combinations for osteoporosis are Lactobacillus acidophilus, Lacticaseibacillus casei, Bifidobacterium longum, Bacillus coagulans, Limosilactobacillus reuteri. Although this conclusion was not based on a systematic review study, it was based on the experiences of expert clinicians in the treatment of osteoporosis and manufacturers of probiotic formulations. In the next step, we divided these strains into several hybrid groups using a mathematical matrix, divided them into seven groups, and examined their preventive effects on osteoporosis in rat. Therefore, we inspected the effectiveness of seven probiotic combinations from five native probiotics (Lactobacillus acidophilus, Limosilactobacillus reuteri, Lacticaseibacillus casei, Bifidobacterium longum, and Bacillus coagulans), as mentioned in Table 1, on BMD, BMC, of global, spine, femur, and tibia on ovariectomized rats.

In this study, combination NO 4, containing L. acidophilus, B. longum, and L. reuteri, is the most influential group on global, spine, and femur BMD. Combination NO 3, containing L. acidophilus, L. casei, and L. reuteri, also significantly affects the BMD of the tibia among the treatment group. According to Kim et al., L. casei considerably improved the tibia BMD in OVX rats [31]. While evaluating the effects of our probiotic combinations on femur, spine, global, and tibia, we indicated that particular combination had the most influence on the special bone type. It was evident that combination NO 4 had the most significant increasing effect on global BMC. Also, combination NO 1 (comprising L. acidophilus, L. casei, and B. longum), NO 6 (containing L. casei, B. longum, and Bacillus coagulans), NO 7 (containing L. casei, L. reuteri, and B. longum), and NO 4 had the most considerable raising effect on spine BMC.

What can be deduced from this study is that L. acidophilus is a common strain in the most effective combination sets tested to improve both BMC and BMC. Our previous study reported that L. acidophilus was more effective in the treatment groups in the case of global, spine, and femur BMD than the OVX untreated group [3]. Some studies reported that L. reuteri might decrease fracture, increase BMD, BMC, and trabecular number and thickness and weaken the trabecular space of the vertebrae and femurs. Inflammation may be the leading cause of abnormal bone regeneration and the onset of bone loss. Some studies have shown that increased inflammatory cytokines are associated with osteoclastic bone resorption, low bone mineral density (BMD), elevated bone resorption, and increased fracture risk. Therefore, blockade of the pro-inflammatory cytokine levels due to intake of probiotic supplements leads to a decrease in bone resorption [32, 33]. We also observed that this strain significantly improved global BMD, BMC, femur BMD, and BMC [3].

Also, B. longum is present in many effective combinations of this study, especially NO 4. Under our previous studies, B. longum could successfully affect femur BMD and BMC. The impact of B. longum on bone density, bone mineral content, bone remodeling, bone structure, and osteoclast/osteoblast gene expression markers was previously evaluated in the OVX rat model for 16 weeks and improved their bone density, trabecular number, thickness, and femoral strength. B. longum likewise reduces levels of serum C-terminal telopeptide [34].

The current study disclosed an apparent synergistic effect between Lactobacillus species and B. longum. Evidence studies in preclinical suggest that dual colonization of Lactobacillus sp. and Bifidobacterium sp. ameliorated severe diarrhea and synergistically decreased virus shedding titers probably because of modulating mucosal and systemic innate and adaptive immunity [35]. This synergistic effect may also be conceivable for the treatment and prevention of osteoporosis. Also, Lactobacillus sp. and Bifidobacterium sp. combination synergistically alleviated immobilization stress and anxiety behaviors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, brain-derived neurotrophic factor (BDNF) expression, tumor necrosis factor (TNF)-α, interleukin (IL)- 6, and lipopolysaccharide levels via maintaining of the gut immune responses and microbiota arrangement [36]. Additionally, these strains repressed the expression of NF-κB activation and TNF-α. Some recent studies indicated such an effect on bone mineralization when naturally combined Lactobacillus sp. and Bifidobacterium sp., attributed to changes in gut microbiota and ecology [37]. Most studies that have evaluated the effectiveness of probiotic compounds include Lactobacillus sp. In some studies, however, Bifidobacterium sp. was used in combination with Lactobacillus. This indicates the greater effectiveness of lactobacilli sp. in a mixture. Bifidobacterium sp. was used in most studies that did not show a more significant effect of the probiotic combinations, indicating that the therapeutic effect of Bifidobacterium sp. may be repressed when other species exist in a multivalent probiotic mixture. Among the studies in which the mixture was not more effective, all contained one or more species of lactobacilli along with Bifidobacterium sp. and several other genera. This reduction in effect indicates that sometimes the high diversity of strains in a probiotic combination diminishes the effectiveness of a multivariate probiotic. These diverse strains appear to constrain each other in the body environment, possibly due to the secretion of antagonistic agents or struggle for nutrients or receptors in the gastrointestinal tract. Our previous study revealed that oral administration of B. longum or Lactobacillus strains declines BMD and BMC criteria induced thorough ovariectomizing in response to hormone deficiency. However, the mixture of these probiotics was more effective in osteoporotic rats at all levels of BMD and BMC. This study’s results indicated that probiotic’s ameliorative effect on bone loss in ovariectomized rat model is strain-specific. The consequences have also revealed a discrepancy concerning the influence of these different probiotic treatments on releasing plasma levels of hormones. It seems that the L. acidophilus strain, combined with L. casei and B. longum strains, regulated immune systems, increased calcium absorption, and even ameliorated hormone levels under postmenopausal like osteoporosis conditions. The role of B. longum in the mixture of other strains can be elucidated by its anti-inflammatory features. The action of B. longum depends on the inflammatory state of the intestine, and it induces an anti-inflammatory response in inflammatory bowel disease. The anti-inflammatory properties of B. longum may be due to its capability to prevent the binding of pathogens to epithelial cells. Considering the functional specificity of each strain at the peripheral and central levels, all these data demonstrate that CNS function is improved by a mixture of these two or three probiotics (L. Acidophilus, B. Longum, and L. casei). This combination of probiotics is feasible and acceptable to people worldwide with no severe side effects. Although probiotic nutraceutical products have been extensively used for a long time, the use of probiotics to improve general health is increasing. For example, in a recent data network survey, approximately 40% of people indicated that in 2018 they used probiotics in various forms. Probiotic combinations are easy to process, formulate, commercialize, and prescribe in commercial conditions. Three effective probiotic strains in this study are available in various dairy products and other human fermented foods. They can be easily isolated and purified using conventional microbiological methods. Because of the growing interest in probiotics and their association with osteoporosis and bone loss, it is interesting to analyze the cost-effectiveness ratio of this potential intervention for osteoporosis.

However, the cost-effectiveness ratio of probiotic combinations for osteoporosis has not been estimated in the literature. Therefore, evaluating the cost-effectiveness of using probiotic combinations to prevent osteoporosis in postmenopausal women can lead to valuable results.

The limitation of our study was that we did not detect the changes of related metabolites of intestinal flora, metabolic indexes of bacterial flora and bone related pathway proteins, bone remodeling related indexes, and bone microstructure.

Our observations revealed that probiotic combinations could be used for bone formation improvement, bone resorption reduction, and microstructure of femur changing. Among seven combined effective probiotics, a combination containing L. acidophilus, B. longum, and L. reuteri is the most influential group in ovariectomized osteoporotic rat. This combination synergistically increased the level of BMC and BMD together with calcium absorption, osteoblast activity, and vitamin D concentration that cause elevated bone health. Further clinical studies are needed to ensure that these reports are qualified for human beings.