Effect of vitamin K 2 on bone mineral density and serum cathepsin K in female osteoporosis patients

Purpose: To investigate the influence of vitamin K2 on bone mineral density, bone metabolism and serum tissue protease K (cathepsin K) in female patients with osteoporosis. Method: A total of 210 osteoporosis patients in Affiliated Hospital of Nanjing University of Chinese Medicine who met the inclusion criteria were selected from January 2017 to January 2018. The patients were randomly divided into vitamin K2 group, strontium renate group and blank control group (70 patients/group). Strontium ranelate group was orally given 2 g of strontium ranelate daily, while vitamin K2 group received 15 mg of Gulikang capsule 3 times a day. Bone mineral density (BMD) and serum osteocalcin (BGP), β-collagen degradation product (β-crosslaps), type I procollagen amino terminal propeptide (PINP), cathepsin K (cathe K) and TRAP were measured prior to drug treatment, and six months after surgery, using standard procedures. Results: Relative to the blank control, hip and lumbar spine density of vitamin K2 and strontium ranelate groups increased to varying degrees. Strontium ranelate group had significantly higher bone mineral density (BMD) than any other groups (p < 0.05), and also had the lowest osteoclast activity (β-crosslaps and TRAP) and the highest osteogenic activity (BGP and PINP). On the other hand, osteoclast and osteogenic activities increased significantly (p < 0.05) in the vitamin K2 group. Conclusion: Appropriate vitamin K2 treatment improves BMD in the hip and waist of women with osteoporosis by promoting osteogenic activity, and by reducing osteoclast activity and cathepsin K expression.


INTRODUCTION
Post-menopausal osteoporosis (PMO) refers to changes in bone metabolism caused by decreased estrogen levels, resulting in increased loss of osteoclasts and trabecular bone.It causes bone fragility and bone fractures.At present, there are about 200 million people who suffer from osteoporosis in the world, and its incidence is now ranked 7 th in the list of common diseases in the world [1].Osteoporosis is a frequently-occurring disease, and it is marked by decreased bone mass and altered bone structure, which lead to increased bone fragility and ultimately increased risk of bone fracture [2,3].
Due to aging populations, the prevalence of osteoporosis is expected to significantly increase in the future [4,5].Osteoporosis occurs mostly in women and elderly men [6].Studies have found that about 25 % of trabecular bone and 3 % of cortical bone in adults undergo a dynamic balancing process of osteoclastic bone resorption and osteoblastic bone formation [7].When the balance in bone remodeling is impaired, osteoporosis results because bone resorption becomes greater than bone formation [8].Therefore, the aim of therapy for osteoporosis is to minimize risk of first or secondary fractures.At present, the antiosteoporosis drugs in use are agents that inhibit bone resorption and increase bone formation.They include parathyroid hormone [9] and bisphosphonates [10].In recent years, studies have indicated that vitamin K 2 is crucial in many aspects of bone metabolism.Vitamin K 2 ameliorates imbalance in bone tissue metabolism through regulation of bone biogenesis and inhibition of the rate of bone resorption [11].
Cathepsin K is expressed mainly in osteoclasts, and it is widely distributed in bone resorption surfaces, transcellular vesicles and lysosomes [12].It is a key protease for bone resorption.Increased expression of cathepsin K in osteoclasts leads to increased osteoclast metabolism, accelerated bone resorption, and ultimately osteoporosis [13].In view of this, it can be hypothesized that vitamin K 2 regulates bone metabolism in osteoporotic women by influencing cathepsin K. Therefore, the aim of this research was to study the influence of vitamin K 2 on cathepsin K, BMD and bone metabolism in patients with osteoporosis.

EXPERIMENTAL Clinical profile of patients
Osteoporotic patients (210 cases) who visited our hospital from January 2017 to January 2018, and who satisfied the inclusion requirements were used as study subjects.These patients were randomly assigned to vitamin K 2 group, strontium renate group and blank control group.The selected patients were 50 -80 years old, with a mean age of 64.07 ± 9.63 years.The average age at menopause was 48.69 ± 7.39 years, the average height was 158.82 ± 7.45 cm, and the average weight was 60.03 ± 12.39 kg.All 210 patients completed follow-up after 6 months of drug treatment.Age and menopausal time were comparable among the three groups (p > 0.05).The study received approval from the Ethical Committee of Affiliated Hospital of Nanjing University of Chinese Medicine (approval no.20180726), and was carried out according to the Declaration of Helsinki promulgated in 1964 as amended in 1996 [14].All the patients signed informed consent.

Inclusion criteria
The recommended diagnostic criterion for OP was reduction of dual-energy X-ray bone density equal or higher than 2.5 standard difference [15].

Exclusion criteria
(1) Patients with osteomalacia, calcium and vitamin D deficiency and renal tubular acidosis; (2) patients with primary and secondary hyperthyroidism; (3) patients with bone metastasis of malignant tumor; (4) multiple myeloma patients; (5) patients who had spinal hemangioma; (6) patients with suppurative myelitis; (7) patients with motor disease, chronic medical diseases and endocrine diseases caused by secondary osteoporosis, and (8) patients on estrogen, heparin, corticosteroids, bisphosphonates and other drugs related to osteoporosis.

Treatments
All patients were subjected to conditioned lifestyles, and were required to take one piece of calcium carbonate D 3 chewable tablet as a basic measure.The vitamin K 2 group was given Gulikang capsule (Fuji Capsule Co., Ltd.Shibakawa Plant) treatment 3 times a day (15 mg each time).Patients in the strontium ranelate group were orally administered 2 g of strontium ranelate (France Servier), daily.The 3 groups were followed up for 6 months after treatment.

Therapeutic indices
Fasting venous blood was collected before treatment, and 6 months after treatment, and serum samples were separated at room temperature.The levels of bone gla protein (BGP), beta-collagen degradation product (βcrosslaps), and PINP were determined using Roche Cobase 601 automatic electrochemiluminescence analyzer (Royce, Switzerland).Serum cathepsin K (cathe K) and TRAP were assayed with ELISA (R & D Corporation, US).

Determination of BMD
This was carried out with dual energy x-ray absorptiometry (DXA) and dual energy x-ray bone density meter (GE-Lunar, GE) before, and 6 months after treatment.The BMD of lumbar vertebrae L 2-4 , femoral neck, greater trochanter, and total hip were recorded.

Statistical analysis
Data were analyzed was done with SPSS 19.0 software package.Numeric data are expressed as mean ± SD and were analyzed with Student's t-test.Count data was analyzed using χ 2 test.Correlation analysis was done with Spearman correlation.Statistical significance was fixed at p < 0.05.

Bone turnover index BGP, β-crosslaps, PINP, TRAP and Cathe K
As shown in Table 1, after 6 months of drug treatment, the serum levels of BGP, β-crossLaps, PINP, TRAP and cathe K were changed to different degrees, relative to their levels before treatment.Relative to the blank control group, changes in bone metabolism and cathe K were more obvious in the vitamin K2 group and the strontium ranelate group than in control (p < 0.05).Changes in bone metabolism in the patients given strontium ranelate group were more obvious than those in the vitamin K 2 group, while changes in cathe K were markedly higher in vitamin K 2 group than in the group that received strontium ranelate (p < 0.05).

Changes in bone mineral density
There were no significant differences in BMDs of lumbar vertebrae (L 2-L 4 ), femoral neck, greater trochanter and total hip before treatment (p > 0.05).However, after 6 months of drug treatment, there were significant increases in BMDs of femoral neck, lumbar vertebrae, greater trochanter and total hip, when compared with their values before treatment (p > 0.05).Compared with the blank control group, the bone mineral densities of these tissues were significantly increased in the vitamin K 2 and strontium ranelate groups, but the increases were higher in the strontium ranelate group than in the vitamin K 2 group (p > 0.05).These results (Table 2) showed that vitamin K 2 enhanced the treatment of osteoporosis, but its effect was not as good as that of strontium ranelate.

DISCUSSION
In this study, 210 women with osteoporosis were selected as subjects, and they were treated with vitamin K 2 or strontium ranelate.After 6 months of treatment, the levels of indicators of changes in BMD and bone metabolism i.e.BGP, β-crossLaps, PINP, TRAP and cathe K in lumbar vertebrae (L 2-4 ) and femoral neck, greater trochanter and total hip, were measured in the three groups.The results showed that strontium ranelate and vitamin K 2 had significant therapeutic effects on osteoporosis.Both treatments significantly increased BMDs of femoral neck, lumbar vertebrae, greater trochanter and total hip.Moreover, the treatments reduced bone resorption activity (marked by β-crossLaps, TRAP, and cathe K), and promoted osteogenic activity (marked by BGP and PINP).These results indicate that strontium ranelate and vitamin K 2 are effective in the treatment of osteoporosis.
Barium ranelate is an anti-osteoporosis drug that acts by reducing bone resorption and increasing bone formation.It is used for treating osteoporosis and for reducing susceptibility to fractures, while simultaneously enhancing bone density and strength.

Study limitations
This study has some limitations.Firstly, the number of subjects used was small, and the follow-up time (6 months) was too short for any meaningful comparison of the effects of the two treatments.Secondly, the dose of the drugs used may not have been appropriate, and this could have influenced the outcome of the study.These limitations should be addressed in subsequent studies.

CONCLUSION
The results obtained in this study clearly show that vitamin K 2 increases bone density, promotes osteogenesis and inhibits bone fracture.It is more effective in treating osteoporosis than strontium ranelate, and it can be taken for a long time.It inhibits the expressions of β-crossLaps and TRAP, promotes the expressions of BGP and PINP, and inhibits the activity of osteoclasts by inhibiting the synthesis of cathe K. Thus, vitamin K 2 is a safe, effective and suitable drug for protection from, and treatment of osteoporosis in the elderly.

Table 1 :
Serum levels of BGP, β-crossLaps, PINP, TRAP, and Cathe K before and after 6 months of treatment (n = 70) * P < 0.05, relative to value before treatment in the same groups, # p < 0.05, relative to blank control group