Prevention of bone loss by using Lactobacillus-fermented milk products in a rat model of glucocorticoid-induced secondary osteoporosis

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Abstract

Secondary osteoporosis is a common side effect of glucocorticoid treatments. The anti-osteoporotic role of two fermented milk products (FMPs), produced with Lactobacillus plantarum A41 and Lactobacillus fermentum SRK414, in alleviating secondary osteoporotic symptoms induced by dexamethasone (Dex) injection in rats was investigated. The two FMPs could prevent Dex-induced bone loss in rats. The two FMPs normalised altered gene expression levels of bone metabolism-related markers (osteoprotegerin, osteocalcin, bone sialoprotein, receptor activator of nuclear factor kappa-B ligand, and tartrate-resistant acid phosphatase). Furthermore, they regulated gene expression levels of bone morphogenetic protein 2-related and cell apoptosis markers in the tibia and calcium metabolism-related markers in the ileum. Additionally, these treatments modulated faecal bacterial populations by increasing Lactobacillus colonies. FMPs fermented by L. plantarum A41 and L. fermentum SRK414 were capable of preventing glucocorticoid-induced secondary osteoporosis via regulation of intestinal microbiota and gene expression levels of bone cell apoptosis related markers.

Introduction

Osteoporosis is a metabolic skeletal disease, characterised by a systemic decrease of bone mass and impairment of the bone microarchitecture, which increases the risk of bone fracture (Riggs & Melton, 1995). The number of people with osteoporosis is increasing owing to the growing aging population worldwide (Kim et al., 2016; Si, Winzenberg, Jiang, Chen, & Palmer, 2015). Osteoporosis results from an imbalance in bone remodelling, which is regulated by the activities of organic bone matrix-producing osteoblasts and bone-resorbing osteoclasts (Karsenty, Kronenberg, & Settembre, 2009); higher activity of osteoclasts leads to increased bone resorption, causing bone loss, unbalanced bone remodelling, and ultimately, osteoporosis.

The most common form of osteoporosis is primary osteoporosis, which includes postmenopausal osteoporosis (owing to oestrogen deficiency) and senile osteoporosis (owing to aging) (Jilka et al., 1992). Secondary osteoporosis results from secondary causes, such as endocrine disorders, malnutrition, autoimmune disease, renal disease, and side effects of therapeutic drugs such as glucocorticoids (GCs) (Colangelo, Biamonte, Pepe, Cipriani, & Minisola, 2019). The incidence of secondary osteoporosis is difficult to discern, but it has been suggested to occur mostly in two-thirds of men and one-fifth of postmenopausal women with osteoporosis (Collins, Rios-Arce, Schepper, Parameswaran, & McCabe, 2017). GCs are a highly effective treatment for inflammatory and autoimmune related diseases, but their adverse effects limit their use (Henneicke, Gasparini, Brennan-Speranza, Zhou, & Seibel, 2014). GC-induced osteoporosis is the most common cause of secondary osteoporosis; long-term use of GCs has the potential to cause a decrease in bone mineral density (BMD) and bone strength. Several guidelines for treating GC-induced osteoporosis are available, but the proportion of research on secondary osteoporosis remains low (Briot & Roux, 2015). Thus, the development of alternative therapies to counteract GC-induced osteoporosis is imperative.

Lately, the role of the intestinal microbiota in regulating bone health has received increasing attention. The commensal gut microbiota in human intestine performs useful functions by modulating both host metabolism and immune status (Rooks & Garrett, 2016). The composition of gut microbiota is modulated by factors such as diet, antibiotics, prebiotics, and probiotics. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer health benefits on the host by altering the gut microbiota composition (Hill et al., 2014). In addition, the gut microbiota composition can be modulated by fermented milk products (FMPs); dairy products fermented by some lactic acid bacterial strains. Several studies have shown that some probiotic strains exert anti-osteoporotic effects in a rat model of ovariectomy-induced postmenopausal primary osteoporosis (Britton et al., 2014; Parvaneh et al., 2015). Similarly, products fermented by Lactobacillus strains exert bone-protecting effects in postmenopausal women and ovariectomised animals (Kim et al., 2009; Narva, Nevala, Poussa, & Korpela, 2004). FMPs are known to contain calcium, phosphorus, protein, and micronutrients, which play a critical role in bone homeostasis (Rizzoli & Biver, 2018). However, limited knowledge exists on the correlation between probiotics and secondary osteoporosis. Zhang et al. (2015) have demonstrated the osteoprotective effect of Lactobacillus reuteri strain on a mice model of type 1 diabetic, which is one of the categories considered as secondary osteoporosis. Since there are various medications and other causes that can induce secondary osteoporosis, alternative methods of preventing such a disease are needed (Mazziotti, Canalis, & Giustina, 2010).

From 485 lactic acid bacteria (LAB) strains, we previously screened and identified two-Lactobacillus strains, Lactobacillus plantarum A41 and Lactobacillus fermentum SRK414, based on their probiotic properties and anti-osteoporotic effects on dexamethasone (Dex)-treated MC3T3-E1 osteoblastic cells (Lee & Kim, 2019). These strains are capable of significantly upregulating the mRNA expression of genes related to bone formation such as osteocalcin (OCN), bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), and thus, were selected for the FMP fermentation process and in vivo trial of secondary osteoporosis.

Here, we examined the effects of the L. plantarum A41 and L. fermentum SRK414 FMPs in a rat model of secondary osteoporosis that showed decreased bone metabolism owing to Dex injection.

Section snippets

Experimental animals

The experiment was performed using protocols approved by the Korea University Institutional Animal Care & Use Committee, South Korea (KUIACUC-2018-14). Forty-two 8-week-old female Wistar rats (weight range, 180–190 g) were obtained from Samtako Bio-Korea Inc. (Seoul, South Korea). The rats were housed in an animal room at the Gyerim Experimental Animal Resource Centre, Korea University, with controlled environmental conditions (temperature, 23–25 °C; relative humidity, 50–55%; 12 h day/night

Effect of Lactobacillus FMPs on growth performance in rats with Dex-induced secondary osteoporosis

The effects of Lactobacillus FMPs on growth performance in rats with Dex-induced secondary osteoporosis are shown in Table 1. Intramuscular Dex injection decreased (P < 0.05) the final body weight in all experimental groups compared with the Normal group. However, the Dex-induced decrease in body weight was partially prevented in the FMP-supplemented groups (A41 and SRK414) and the Drug group. The feed intake was also lowered in the Dex group (P < 0.05) at the end of the experimental period.

Discussion

Glucocorticoids are known to be the most frequently used anti-inflammatory and immunosuppressive drugs for the treatment of diseases such as cancer, autoimmune disorders, and inflammation (Rhen & Cidlowski, 2005). However, GC therapy is known to cause osteoporosis through inhibition of intestinal Ca absorption, activation of apoptosis in bone-forming osteoblasts, and increase in the activity of osteoclasts (Chang et al., 2009; Lafage-Proust, Boudignon, & Thomas, 2003). Many studies have shown

Conclusion

In the present study, we have demonstrated the anti-osteoporotic effects of two previously screened Lactobacillus strains on a rat model of GC-induced secondary osteoporosis. Both L. plantarum A41 and L. fermentum SRK414 FMPs were capable of preventing Dex-induced bone loss via alterations in the mRNA expression levels of bone metabolism-related markers OPG, OCN, BSP, ALP, and RANKL. Additionally, by confirming the mRNA expression levels of major related genes, we further discovered that the

Acknowledgements

This work was supported by the High Value-Added Food Technology Development Program of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET), the Ministry for Food, Agriculture, Forestry and Fisheries of Republic of Korea (117051-03-1-HD020), and a Korea University grant.

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