J Korean Neurosurg Soc.  2022 Mar;65(2):204-214. 10.3340/jkns.2021.0098.

The Effect of Genetically Modified Lactobacillus plantarum Carrying Bone Morphogenetic Protein 2 Gene on an Ovariectomized Rat

• Jin ES
• Kim JY
• Yang JM
• Kim JS
• Min J
• Jeon SR
• Choi KH
• Moon GS
• Jeong JH

• KMID: 2527177
• DOI: http://doi.org/10.3340/jkns.2021.0098

Abstract

Objective
: Osteoporosis result from age-related decline in the number of osteoblast progenitors in the bone marrow. Probiotics have beneficial effects on the host, when administered in appropriate amounts. This study investigated the effects of probiotics expressing specific genes, especially the effects of genetically modified bone morphogenetic protein (BMP)-2-expressing Lactobacillus plantarum CJNU 3003 (LP) on ovariectomized rats.
Methods
: Twenty-eight female Wistar rats (250–300 g, 12 weeks old) were divided into four groups : the sham (control), the ovariectomy (OVX)-induced osteoporosis group (OVX), the OVX and LP (OVX/LP), OVX and genetically modified BMP-2-expressing LP (OVX/LP with BMP) groups. The three groups underwent bilateral OVX and two of these groups were administered two different types of LP via oral gavage daily. At 16 weeks post-OVX, blood was collected from the heart and the bilateral tibiae were extracted and were scanned by ex-vivo micro-computed tomography and stained with hematoxylin-and-eosin (H&E) and Masson’s trichrome stain for pathological assessment. The serum levels of osteocalcin (OC), rat C-telopeptide of type I collagen (CTX-I), BMP-2, and receptor activator of nuclear factor-ĸB ligand (RANKL) were measured.
Results
: The 3D-micro-computed tomography images showed that the trabecular structure in the OVX/LP with BMP group was maintained compared with OVX and OVX/LP groups. No significant differences were detected in trabecular thickness (Tb.Th) between control and OVX/LP with BMP groups (p>0.05). Furthermore, a tendency toward increased BMD, trabecular bone volume, Tb.Th, and trabecular number and decreased trabecular separation was found in rats in the OVX/LP with BMP groups when compared with the OVX and OVX/LP groups (p>0.05). The H&E and Masson’s trichrome stained sections showed a thicker trabecular bone in the OVX/LP with BMP group compared with the OVX and OVX/LP groups. There was no difference in serum levels of OC, CTX and RANKL control and OVX/LP with BMP groups (p>0.05). In contrast, significant differences were found in OC and CTX-1 levels between the OVX and OVX/LP with BMP groups (p<0.05).
Conclusion
: Our results showed that the expression of genetically modified BMP-2 showed inhibition effect for bone loss in a rat model of osteoporosis.

Keyword

Probiotics; Osteoporosis; Models, animal; Gene editing; Bone morphogenetic protein 2

Figure

• 

  Fig. 1. Confirmation of recombinant Lactobacillus plantarum CJNU 3003 (LP; pLR5cat-PSBMP2) by polymerase chain reaction (PCR). Escherichia coli DH5α (pLR5cat-PSBMP2) as positive control for PCR template (a); LP (pLR5cat-PSBMP2) candidate (b). Lane 1, 4 : PSamyF and PSamyR primer set, Lane 2, 5 : bone morphogenetic protein (BMP)-2 factor and BMP-2 receptors primer set, Lane 3, 6 : PSamyF-BMP2R primer set, Lane M : size marker (MG 1 kb plus DNa ladder marker; MGmed Co., Seoul, Korea).

• 

  Fig. 2. Graphic analysis of body weight. In all groups, the mean body weight increased over 20 weeks after ovariectomy-induced osteoporosis group (OVX). Weights of rats in the OVX, OVX and Lactobacilus plantarium CJNU 3003 (LP) feeding group (OVX/LP), OVX and genetically modified bone morphogenetic protein (BMP)-2-expressing LP feeding group (OVX/LP with BMP) were higher than in the control group (sham group). Significantly, the body weight in the OVX/LP, OVX/LP with BMP group was slightly lower than that in the OVX group. All data are expressed as the mean±standard error of mean. Two-way aNOVa, bonferroni post-tests *p<0.05, †p<0.01, ‡p<0.001 vs. control.

• 

  Fig. 3. A representative micro-computed tomography image. In spite of osteoporotic changes in rats in the ovariectomy-induced osteoporosis group (OVX), OVX and Lactobacilus plantarium CJNU 3003 (LP) feeding group (OVX/LP), OVX and genetically modified bone morphogenetic protein (BMP)-2-expressing LP feeding group (OVX/LP with BMP), the trabecular structure was highly preserved in the OVX/LP with BMP group than in OVX and OVX/LP groups. Control, sham group.

• 

  Fig. 4. Graphic analysis of micro-computed tomography evaluation. The control group (sham group) had the highest values of bone mineral density (BMD), trabecular bone volume (BV/TV), trabecular thickness (Tb. Th), and trabecular number (Tb.N) and the lowest values of trabecular spacing (Tb.Sp). The differences in Tb.Th between control and ovariectomy (OVX) and genetically modified bone morphogenetic protein (BMP)-2-expressing Lactobacilus plantarium CJNU 3003 (LP) feeding group (OVX/LP with BMP) were not significant. All data are expressed as the mean±standard deviation. *p<0.05, †p<0.01, ‡p<0.001 vs. control.

• 

  Fig. 5. Images of (A) hematoxylin and eosin (H&E) and (B) Masson’s trichrome staining of tibia. The images show loss of bony trabeculae and increased adipose tissue in the bone marrow of rats in the ovariectomy-induced osteoporosis group (OVX), OVX and Lactobacilus plantarium CJNU 3003 (LP) feeding group (OVX/LP), OVX and genetically modified bone morphogenetic protein (BMP)-2-expressing LP feeding group (OVX/LP with BMP). However, there are more preservation of the trabecular formation in the OVX/LP with BMP group compared to the OVX and OVX/LP group. Control, sham group.

• 

  Fig. 6. Changes in the levels of serum markers. A : The serum level of bone morphogenetic protein (BMP)-2 was higher in the control group (sham group) than in the other three groups, with the lowest level observed in the ovariectomy-induced osteoporosis group (OVX) group (p<0.05). There is no significant difference in serum biomarker levels between the OVX, OVX and Lactobacilus plantarium CJNU 3003 (LP) feeding group (OVX/LP), and OVX and genetically modified BMP-2-expressing LP feeding group (OVX/LP with BMP) (p>0.05). B-D : Serum levels of osteocalcin (OC), C-telopeptide of type I collagen (CTX-I), and receptor activator of nuclear factor-ĸB ligand (RaNKL) were the highest in the OVX group, with significant differences between OVX and control group. However, there is no difference between control and OVX/LP with BMP (p>0.05). In contrast, significant differences in OC and CTX-1 existed between the OVX and OVX/LP with BMP groups (p<0.05). All data are expressed as the mean±standard deviation. *p<0.05, †p<0.01, ‡p<0.001 vs. control, §p<0.05, =p<0.01, ¶p<0.001 vs. OVX.

Reference

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