The role of Bacillus acidophilus in osteoporosis and its roles in proliferation and differentiation

Abstract Background Osteoporosis is one of the most closely related diseases associated with the elderly. In recent years, the studies found that gut microbiota can cause osteoporosis. We evaluated the role of Bacillus acidophilus in osteoporosis and its roles in proliferation and differentiation. Methods We selected 5 healthy people and 10 osteoporosis patients and analyzed their level of 25‐hydroxyvitamin D and procollagen type I N‐terminal peptide (PINP), the characteristic of gut microbiota. The effect of lactobacillus acidophilus and Lactobacillus rhamnosus supernatant and butanoic acids on proliferation, differentiation, and maturity of osteoblasts MC3T3‐E1 and osteoclasts RAW 264.7 cells and the activity of alkaline phosphatase, concentration of osteocalcin, and the expression of RUNX2, RANK, NFATc1, cathepsin K, DC‐STAMP, OSCAR, WNT2, and CTNNB1 were measured in the above cell lines. Results The diversity of gut microbiota in osteoporosis patients is decreased and imbalanced with lower abundance of lactobacillus and butyric acid bacteria; meanwhile, 25‐hydroxyvitamin D and PINP of osteoporosis patient were significantly lower than the normal group. The proliferation, differentiation, and maturity of MC3T3‐E1 cells were stimulated; the activity of alkaline phosphatase, concentration of osteocalcin, and the expression of RUNX2, NFATc1, cathepsin K, DC‐STAMP, OSCAR, WNT2, and CTNNB1 were improved by supernatant of lactobacillus acidophilus, Lactobacillus rhamnosus and butanoic acids; however, the proliferation, differentiation, maturity, and the expression of RANK, NFATc1, cathepsin K, DC‐STAMP, OSCAR, WNT2, and CTNNB1 in RAW 264.7 cells were suppressed. Conclusions The lactobacillus acidophilus and Lactobacillus rhamnosus supernatant could stimulate the proliferation, differentiation, and maturation of osteoblasts; the production of butyric acid may be the potential mechanism.


| INTRODUC TI ON
As a mammal, humans have an incredibly large number of microbes living on and inside our bodies and a complex community of symbiotic bacteria. 1 These microorganisms play an important role in the physiological activities of the human body. The largest microecosystem in the human body is the intestinal tract. 2 In recent years, increasing evidence has accumulated, suggesting that the human gut microbiota can be thought of as a very important organ acquired after birth. 3 New attention has been directed at the relationship between the gut microbiota and human health. The interaction between the gut microbiota and the host is mutualistic, as the host can provide a suitable environment and nutrients to support bacterial growth while the gut microbiota plays important roles in food digestion, the production of vitamins and other nutrients, and increasing resistance against invasion by foreign pathogens. 4,5 In addition to these important roles, the microbiota can also affect many physiological functions of the human body. Under normal circumstances, the intestinal microecosystem remains relatively stable, and this balance is beneficial to the human body. However, a variety of factors (such as drugs, alcoholism, mental factors, gastrointestinal surgery, radiation therapy, and aging) can alter this balance, resulting in the development of a variety of diseases, such as multiple enteritis, diabetes, asthma, obesity, osteoporosis, and metabolic syndrome. [6][7][8][9][10] Osteoporosis is a disease intimately linked to aging. 11 It is a common bone disease characterized by decreased bone mass and bone degeneration. Recent studies have shown that the gut microbiota is associated with decreased bone mass and the pathogenesis of osteoporosis. 12 These microorganisms may alter the relative activities of osteoclasts and osteoblasts through different pathways, such as metabolite production and altering of the host metabolism and immune system, both of which can affect bone metabolism.
Therefore, the goals of this study were to analyze the changes in the gut microbiota in osteoporosis patients and to study the effects of the metabolites of two probiotics, Lactobacillus acidophilus and Lactobacillus rhamnosus (LGG), on an osteoblast precursor cell line (MC3T3-E1) and on an osteoclast precursor cell line (RAW 264.7). By studying the relationship between the gut microbiota and osteoporosis and investigating the mechanism by which the gut microbiota affects bone metabolism from the perspective of osteoblast-mediated bone formation and osteoclast-mediated bone resorption, this study could lay a theoretical foundation for further investigation into these topics and provide data to support the use of probiotics as a clinical intervention for osteoporosis.

Age
The diagnostic criteria for osteoporosis were based on T-scores ≤−2.5 in any of the following sites: lumbar vertebrae, femoral neck, or total hip.

| Fecal DNA extraction and testing
Stool specimens were collected from the patients in the osteoporosis and healthy control groups using a sterile specimen container.

| Cell culture
Mice osteoblasts MC3T3-E1 were purchased from the Tianjin

| Bacterial culture and supernatant extraction
Cryogenic vials containing the lactobacillus acidophilus (LABS) and Lactobacillus rhamnosus (LGG) solution stored in a liquid nitrogen tank were removed and completely thawed at room temperature.
One milliliter of the bacterial solution was pipetted into 100 mL of

| Determination of ALP activity and OCN
MC3T3-E1 cells were inducted by induction medium which contained vitamin C (50 mg/L) and β-sodium glycerophosphate β-catenin gene (CTNNB1), and receptor activator for nuclear factor-κ B (RANK), and GAPDH was used as the internal reference gene. The quantified results were calculated using the 2 −ΔΔCq method. 13 The full details of the primers used in these experiments are shown in Table 2 2

| Comparison of the general data between the groups
No statistically significant differences were observed between the two groups upon comparison of the biochemical parameters (ALB, ALT, AST, ALP, CREA, UREA, UA, FPG, Ca, P, K, Na, TG, TC, LDL, and PTH) in

| Changes in the gut microbiota of osteoporosis patients
High-throughput sequencing was used to analyze and compare the 16S rRNA sequences present in the gut microbiota of the osteoporosis patients and healthy controls. The structure and characteristic of the gut microbiota in the osteoporosis patients were altered ( Figure 1). Especially, the levels of Lactobacillus and butyric acidproducing bacteria were decreased, and abundance of pathogenic bacteria, such as Clostridium, was increased.

| Effects of LABS, LGG, and sodium butyrate on the proliferation of osteoblasts and osteoclasts
There is a significantly enhanced proliferation of osteoblast    Figure 3C).

| Effects of LABS, LGG, and sodium butyrate on the maturity of osteoblasts and osteoclasts
After

| D ISCUSS I ON
The aging population and age-related bone damage have developed into major public health problems in China. 15 Osteoporosis patients are not only increasing in number but also have increased risk of suffering from additional senile diseases. 16 Osteoporotic fractures in these patients result in difficulties in treatment and care and can also cause many economic and social problems. 17  Lactobacillus is one of the most widely studied probiotics, and the term "lactobacillus" is actually a generic name for any bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. 21 In this study, LABS and LGG, which belong to the  c-fos, which plays an important role in osteoblast and inhibits osteoclast formation, is a part of AP-1. We found that the relative promoter activity of AP-1 was changed with the same tendency. These results confirm that LABS supernatant and butyric acid can promote the differentiation and maturation of osteoblasts and inhibit the proliferation and differentiation of osteoclasts by regulating the expression levels of the above genes.
The experimental results showed that a high concentration of LABS supernatant could stimulate the proliferation, differentiation, and maturation of osteoblasts. The aforementioned effects were not present in cells exposed to LGG supernatant. LABS can produce butyric acid, which can stimulate osteoblast proliferation and differentiation, while LGG does not produce butyric acid. Based on these observations, we speculated that LABS may stimulate osteoblast proliferation, differentiation, and maturation via butyric acid production.