The latest investigations indicated a close relation between gut microbiota composition and bone homeostasis (25) and probiotics in gut-bone signaling (26). Our previous study revealed the supportive role of probiotic live cells in protecting rats from ovariectomy-induced bone loss (17). This study is a novel in which we explored the effects of postbiotics (lysate and supernatant of probiotics) in protecting rats from bone loss induced by ovariectomy. The impacts of postbiotics on various bone compartments have been investigated in the current study. The results showed that Lactobacillus reuteri lysate, Bifidobacterium longum lysate, and Bacillus coagulans lysate increased serum Ca concentration same to the control group. Similar observations have been found for Bifidobacterium longum in our previous study. Ghanem et al. reported that probiotic yogurt enriched with L. reuteri enhanced calcium absorption in growing rats (27). Perez-Conesa et al. explained that Bifidobacterium bifidum and Bifidobacterium longum augmented apparent absorption and apparent calcium retention in weanling rats (28). Yan et al. indicated that dietary supplementation of a Bacillus subtilis based probiotic improves broiler bone traits, most likely through increased calcium intestinal absorption and reduced bone resorption by inhibiting sympathetic activity via the central serotonergic system (29). A study on Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus gasseri reported higher apparent calcium absorption in growing rats and 35% higher bone weight among the postbiotic fed group compared to the control group (30).
In line with our recent study, Lactobacillus acidophilus lysate significantly decreased serum phosphorus compared to the untreated OVX group. Also, we found that Bacillus coagulans supernatant significantly decreased ALP compared to the OVX group, while no significant changes were detected for other postbiotic groups.
In the current study, we also investigated the effects of postbiotics (lysate and supernatant of probiotics) on bone quality (Area, BMC, and BMD). The results revealed that not only live probiotics but postbiotics could considerably improve the global and femur area in OVX rats. In agreement with our previous study, all postbiotics ameliorated the global area. For the femur area, Lactobacillus casei lysate and supernatant, Bifidobacterium longum lysate, Bacillus coagulans lysate, Lactobacillus acidophilus lysate, and supernatant, Lactobacillus reuteri lysate displayed positive effects. No significant improvement had been detected in the spine area in this study.
In contrast to our previous outcomes, Bacillus coagulans lysate and Lactobacillus reuteri supernatant decreased the tibia area, while similar to our previous study, no significant differences were found in other postbiotic supplemented groups. In the case of global BMC, Lactobacillus casei lysate and supernatant, Bacillus coagulans lysate and supernatant, lysate of Bifidobacterium longum and Lactobacillus acidophilus, and Lactobacillus reuteri supernatant significantly increased BMC compared to the OVX group. In comparison with our previous study, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus reuteri revealed similar results. Regarding spine BMC, only Lactobacillus casei supernatant considerably increased BMC, while our previous study on live probiotics showed no progressive effect on spine BMC. Lactobacillus casei and Bacillus coagulans lysate and supernatant, lysate of Bifidobacterium longum and Lactobacillus acidophilus significantly enhanced femur BMC whereas previously, no significant differences were observed. Perez-Conesa et al. recommended that increasing calcium absorption in the distal colon is directly associated with increasing calcium contents of the femur and tibia (28). In agreement with Perez-Conesa et al. study, in the current study, we observed that OVX groups in which postbiotics increased serum calcium concentration (Bifidobacterium longum and Bacillus coagulans-treated groups) had higher femur BMC. In addition, we found Bacillus coagulans supernatant meaningfully enriched tibia BMC. In the case of tibia BMC, similar results were detected for Lactobacillus casei compared to our previous work. Bacillus coagulans supernatant also increased global and spine BMD, while other postbiotics did not show significant changes. Lysate and supernatant of investigated strains like their live forms revealed no positive impact on femur BMD. Lactobacillus casei lysate and supernatant comparable to its live form increased tibia BMD. Similarly, Kim et al. specified that a decreased level of BMD in OVX rats would be significantly improved by administrating Lactobacillus casei 393 from fermented milk (14).
Furthermore, Bacillus coagulans lysate and supernatant, and Lactobacillus reuteri lysate enhanced tibia BMD which was significantly different from their life forms. Recent studies revealed other mechanisms for probiotic effects on bone. Lactobacillus reuteri prevented ovariectomy-induced bone loss via changes in bone marrow CD4 + T cells (31). Lactobacillus casei supplementation repressed osteolysis and the pro-inflammatory state of the macrophages (32). In another work, Lactobacillus reuteri 6475 improved bone health by reducing tumor necrosis factor (TNF) levels and decreasing bone resorption. The results showed an increased bone fracture, BMD, BMC, trabecular number and thickness, and falling trabecular space in both vertebral and femoral bones (33). Parvaneh et al. presented that Bifidobacterium longum treatment augmented BMD, but rather than decreasing bone resorption markers, they observed increased bone formation (34).
Postbiotics are defined as extracellular or intracellular substances produced through the metabolic activity of the microorganism in a different phase of growth and could utilize a favorable effect on the host, directly or indirectly (35). According to the above definition, postbiotics are classified into different classes, including cell-free supernatants, exopolysaccharides, enzymes, cell wall fragments, short-chain fatty acids (SCFA), and bacterial lysates (36). Postbiotics exhibit pleiotropic activities in the human body. The mechanisms of their health benefits are not clearly defined but might be through immunomodulatory effects, antitumor effects, infection prevention, anti-atherosclerotic effects, and autophagy induction (36).
Lactobacillus acidophilus and Lactobacillus casei supernatants have anti-inflammatory and antioxidant effects on intestinal epithelial cells, macrophages, and neutrophils by reducing the secretion of the pro-inflammatory tumor necrosis factor α (TNF-α) cytokine and increasing the secretion of the anti-inflammatory cytokine interleukin 10 (IL-10) (37). Postbiotics originating from Lactobacillus include valuable compounds such as organic acids and bacteriocin, enhancing the growth of lactic acid bacteria (38). Bacillus coagulans isolated fractions (supernatant, cell wall fragments) induced anti-inflammatory cytokine production and promote T helper (Th)2-dependent immune responses (39).
Quach et al. reported that cell culture supernatant (CCS) fraction from L. reuteri 6475 (< 3 kDa) suppressed the differentiation of monocyte/macrophage cell line into osteoclasts (40). In another study, VPP peptide from Lactobacillus helveticus LBK-16H, because of its low bioavailability, did not display preventive activity against ovariectomy-induced bone loss (41). Rahman et al. exhibited that conjugated linoleic acid inhibits osteoclastogenesis by modulating RANKL signaling (42). Chen et al. revealed that the supernatant of Lactobacillus acidophilus and butanoic acids stimulated the proliferation, differentiation, and maturity of osteoblasts MC3T3-E1 cells was, increased the activity of alkaline phosphatase, elevated concentration of osteocalcin, and the expression of RUNX2, WNT2 and CTNNB1 (43).
Butyrate (an SCFA) induces the differentiation of regulatory T cells (Tregs) in the intestine (44). Reports highlight the bone-regulating capacities of Treg cells, describing mechanisms where Treg cells blunt bone resorption, stimulate bone formation by promoting the differentiation of osteoblasts, and are pivotal for parathyroid hormone (PTH)-stimulated bone formation (45). Tyagi et al. reported that oral delivery of Lactobacillus gasseri LGG or butyrate to eugonadal young mice increased trabecular bone volume due to stimulation of bone formation (45).
Postbiotic effectiveness is similar to probiotics, and given that postbiotics do not contain live cells, the risks and side effects associated with their intake is minimize compared to probiotics (36). Postbiotic do not need colonization and could increase the potency of active microorganisms, keep the microorganisms viable and stable in the product at a high dose, improve shelf-life, and simplify packaging and transport (46). Postbiotics can also be used in situations where it is harder to control and maintain production and storage conditions, such as in developing countries (20). The postbiotics used in the current study were originated from five native probiotic strains (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Bifidobacterium longum, and Bacillus coagulans). In most cases, the current study found that postbiotics revealed similar capacities in ameliorating ovariectomy-induced bone loss as much as a probiotic live-cell, which was explored in our earlier study. The results suggest that postbiotic could be used as a substitute for probiotics in preventing bone loss result from estrogen deficiency, but further studies needed to be done to confirm the present study outcomes. Collectively, the data from the current study suggest that the effects of postbiotics on biochemical and bone parameters may depend on the type of individual species that postbiotics originated from, duration of treatment, the bone compartment examined, and the estrogen deficiency model used. More studies need to be done to explore the optimal administrative dose and duration of the specific postbiotics in protecting ovariectomy-induced bone loss in further animal and clinical investigations. Furthermore, identification and characterization of the intracellular and extracellular bioactive molecule(s) produced by bacteria that target bone formation and resorption and their exact mechanisms could help determine the substances that can potentially be used for treating postmenopausal osteoporosis.
The strength of our study is that here, for the first time, we compared 12 different postbiotic treatments obtained from common probiotic strains on ovariectomy-induced bone loss. In the present work, we showed the strain-specific effects of postbiotics and their specific impacts on various bone compartments. As the limitations, in-depth mechanism of postbiotics effects on ameliorating ovariectomy-induced bone loss was not investigated. Characterization of postbiotics could be useful in finding the most effective compounds with bone-sparing effects. Further, in vivo studies and clinical trials are recommended to be conducted to discover the vast aspects of postbiotics therapy on ameliorating bone loss.