Effect of Shengsui Jiangu Capsule on bone conversion in rats with alcoholic osteoporosis

REN Shu-jun, GAO Wei, YU Chang-jiang, LIANG Yan-lin, LI Yuan-feng, TAN Fu-zhu,XU Wei-ming, XU Xi-lin

1. The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China

2. Graduate School of Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China

3. Ningxia Hui Autonomous Region Hospital and Research Institute of Traditional Chinese Medicine, Yinchuan 750021, China

4. The Second Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin 150001, China

Keywords:Shengsui Jiangu Capsule Alcoholic osteoporosis Vitamin D3 Bone transformation Mechanism of action

ABSTRACT

1. Introduction

Alcohol-induced Osteoporosis (AOP) refers to the long-term and large intake of alcohol that disrupts the balance of bone conversion,affects the shaping and reconstruction of bone tissue, destroys the microstructure of bone, and leads to the degeneration and brittleness of bone structure. A secondary peripheral bone turnover disorder that increases the risk of fracture[1,2] . Studies have shown that longterm excessive intake of alcohol can directly affect osteoblasts, or indirectly affect bone mineral density by affecting bone metabolism such as liver and kidney function, vitamin and serum calcium, and increase fracture risk[3]. Bone tissue is continuously shaped and rebuilt through the self-renewal of osteoclasts, a process known as bone conversion[4]. 25 hydroxyvitamin D3(25(OH)D3) is the main storage mode of vitamin D3in vivo, and vitamin nutrition in vivo can be evaluated by detecting the level of 25(OH)D3in serum[5]. In addition, its active metabolite, 1,25 dihydroxyvitamin D3(1,25(OH)2D3), can induce the synthesis of calcanin in vivo,thus promoting the absorption of calcium and phosphorus ions in the intestinal tract and reabsorption by renal tubular cells, which is conducive to the calcification and formation of new bone [6]. Type 1 tropocollagen N - terminal propeptide (P1NP) as first choice reflects the activity of osteoblasts in bone transformation process of bone formation markers, type I collagen cross-linking carboxyl end peptide (beta CTX) is preferred to represent osteoclast activity of bone resorption markers, can has the specificity and sensitivity of the whole body bone formation [7] and bone resorption [8] of the state.

From the "medical science" cut from the Shengsui Jiangu capsule,with kidney tonifying spleen strengthening bone, dampness,removing blood stasis and activating collaterals. This study established a rat model of alcoholic osteoporosis, objective to observe the effects and correlation of Shengsui Jiangu capsule on serum vitamin D3indices (25(OH)D3, 1,25(OH)2D3), bone conversion markers (β-CTX, P1NP) and bone mineral density(BMD) in AOP rats. To explore the possible mechanism of this prescription in the treatment of alcoholic osteoporosis.

2. Materials and methods

2.1 Experimental animals

120 clean-grade male SD rats were provided by the Experimental Center of Heilongjiang University of Chinese Medicine, with an average age of 6 months and weight of (220±20) g. The animal production certificate number was SCXK (Black) 20160103. The Institutional Animal Care and Use Committee of Heilongjiang University of Chinese Medicine approved the animal studies in this survey and followed the procedures in the guidelines for The Care and Use of Experimental Animals (Ministry of Science and Technology, PRC).

2.2 Experimental agents

Shengsui Jiangu capsule (Preparation Room of the First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Specification: 0.35 g/ tablet, Batch NO. : Z20110036;Executive standard: Black Z-ZJ-060-2010); Calcium carbonate tablet (Specification: 600mg/ tablet, Wyeth Pharmaceutical Co.,LTD., Batch No. : H10950029); Alpha D3(Produced by Israel Teva Pharmaceutical Industries Limited, packaged by Kunming Baker Norton Pharmaceutical Co., LTD., Batch No. J20130162); Liquor(Specification: 500ml/ bottle, Beijing Red Star Erguotou 56° clear flavor liquor, Production date: July 27, 2020, Beijing Red Star Co.,LTD., execution standard: GB/T10781.2 (excellent); 0.9% NaCl injection (Specification: 500 mL: 4.5 g (soft bag), Zhejiang Tianrui Pharmaceutical Co., LTD., Batch No. : H33022240).

2.3 Main experimental equipment and reagents

Table top high speed low temperature centrifuge (model:CenLee20R provided by Hunan Xiangli Scientific Instrument Co.,LTD.); Dual-energy X-ray Bone Density Measuring instrument(PLODIGY general (Lunar) Co., LTD); ELISA Kit: (Rat 25(OH)D3ELISA Kit, Article No. : ML028301; Rat 1,25(OH)2D3ELISA kit,article no. Ml028285, Shanghai enzyme-linked biotechnology co.,LTD. Rat β-CTX ELISA Kit, Specification: 96T/48T, Article No.Csb-e12776r; Rat P1NP ELISA Kit, Specification: 96T/48T, Article No. : CSB-E12774R, provided by Wuhan Huamei Bioengineering Co., LTD.)

2.4 Experimental methods

2.4.1 Grouping of experimental animals

The experimental rats provided by the Experimental Animal Center of Heilongjiang University of Chinese Medicine were randomly numbered after 1 week of adaptive feeding, And according to the weight of the random number table method into the model group(alcoholic osteoporosis model group), blank control group, the western medicine control group (calcium carbonate tablet and afar D3control group), traditional Chinese medicine experimental group(Shengsui Jiangu capsule experimental group) four groups, each 30,separate cage feeding.

2.4.2 Experimental animal model

Methods[9-10] the model was made by intragastric administration of chronic liquor: healthy male SD rats were given a dose of 10 mL/ (kg.d) body weight (4.4 g/10 mL alcohol) by intragastric administration of 56° Red Star Erguotou liquor, once a day. Samples were taken 8, 12 and 16 weeks after intervention, and the alcohol dosage was adjusted according to the weight of the rats monitored weekly. After the last successful intragastric administration, fasted and prohibited water for 12 hours. Samples were collected and tested.

2.4.3 Experimental animal intervention methods

Model group: red Star Erguotou 56° liquor was given intragastric administration at a dose of 10 mL·kg-1·d-1(alcohol volume of 4.4 g/10 mL) in the morning and 0.9% NaCl injection in the afternoon,once a day.

Blank control group: equal amount of 0.9% NaCl injection was given intragastric administration once a day in the morning and afternoon.

Western medicine control group: intragastric administration of the same dose of liquor as model group every morning; In the afternoon,calcium carbonate, alpha D3 suspension and 0.9% NaCl injection were prepared by intragastric administration (the dosage standard of calcium carbonate was 85 mg/10 mL/kg, and the dosage of alpha D3was 0.05 μg/10 mL/kg according to the body weight of rats), once a day.

Traditional Chinese medicine experimental group:the experimental group was given the same dose of liquor as the model group every morning. In the afternoon, Shengsui Jiangu capsule and 0.9% NaCl injection were prepared by intragastric administration (the dosage standard was based on the body weight of rats 4.7 g/10 mL/kg),once a day.The volume of intragastric administration of the above groups was 10 mL·kg-1·d-1. During the experiment, the rats were given standard nutritional feed, free food and water, and continuous intragastric administration and medication for 16 weeks. The dosage was adjusted weekly according to the monitored body weight of rats [11].

2.5 Sampling and detection methods

At the end of 8, 12 and 16 weeks after the intervention,experimental rats with fasting for more than 12 h were randomly selected from each group and sacrificed by cervical dislocation,abdominal cavity was opened, abdominal aortic blood was collected,separated in a high speed and low temperature centrifuge and stored for later use, in strict accordance with the instructions of the ELISA kit. Serum levels of vitamin D3(25(OH)D3, 1,25(OH)2D3) and bone conversion markers (β-CTX, P1NP) were determined. After the rats were sacrificed by dislocation of cervical vertebra, the left femur of the rats was removed, the attached tissue was removed, and washed with normal saline. After the rats were wrapped with sterile saline,the bone density of the femur of the rats was measured by dualenergy X-ray bone density measuring instrument.

2.6 Statistical processing

The experimental data were processed by SPSS25.0 software,and the measurement data were expressed in the form of (±s).ANOVA was used for comparison between groups, and P＜0.05 was considered statistically significant. Pearson test was used for correlation analysis.

3. Results

3.1 Effect of Shengsui Jiangu Capsule on bone density of left femur in AOP rats

According to the BMD test results at the end of 8, 12 and 16 weeks after the modeling intervention, the BMD of femur in the model group was significantly lower than that in the blank control group(P＜0.01), indicating that the model of osteoporosis was successfully modeled by using liquor intragastric administration. Compared with the model group, the bone mineral density of femur in the traditional Chinese medicine experimental group was significantly increased(P＜0.01), and was significantly better than that in the Western medicine group (P＜0.05). Are shown in Table 1.

Table 1 Effect of Shengsui Jiangu Capsule on bone density of left femur in AOP rats(n=10, ±s)

Table 1 Effect of Shengsui Jiangu Capsule on bone density of left femur in AOP rats(n=10, ±s)

Note:Compared with blank control group, ▲P＜0.01; Compared with model group, ●P＜0.01; Compared with western medicine control group, ★P＜0.05

Groups8 the weekend12 the weekend16 the weekend blank control group .275±0.0170.273±0.0190.274±0.015 model group 0.223±0.018▲ 0.216±0.014▲ 0.206±0.019▲Western medicine control group 0.236±0.015● 0.239±0.019● 0.246±0.017●Traditional Chinese medicine experimental group 0.248±0.023●★ 0.254±0.016●★ 0.260±0.015●★F 14.5719.9730.92 P＜0.01＜0.01＜0.01

3.2 changes in serum 25(OH)D3, 1,25(OH)2D3, β-ctx and P1NP levels of rats in the experiment

Biochemical test results showed that, compared with blank control group, serum levels of 25(OH)D3, 1,25(OH)2D3and P1NP in model group were significantly decreased (P＜0.01), and serum β-CTX level was significantly increased (P＜0.01), with statistical significance (P＜0.01). These results suggest that high alcohol intake can decrease the level of active vitamin D3and bone formation in the blood of experimental rats, and stimulate the increase of bone resorption index. Compared with model group, the levels of 25(OH)D3, 1,25(OH)2D3and P1NP in western medicine control group and traditional Chinese medicine experimental group were significantly increased (P＜0.01), and the level of β-CT X in serum was significantly decreased (P＜0.01). It is suggested that the two kinds of drug intervention are beneficial to improve the bone conversion ability of experimental rats. Compared with western medicine control group, the improvement degree of serum vitamin D3index and bone conversion index in traditional Chinese medicine experimental group was better than that in western medicine control group (P＜0.05). Are shown in Table 2.

3.3 Correlation of serum vitamin D3 (25(OH)D3, 1,25(OH)2D3)with bone turnover index (P1NP and β-CTX) and bone mineral density in rats with alcoholic osteoporosis

Pearson correlation test results showed that serum vitamin D3levels (25(OH)D3, 1,25(OH)2D3) were positively correlated with serum bone formation marker P1NP and bone mineral density, and negatively correlated with serum bone resorption marker β-CTX level in rats with alcoholic osteoporosis.Are shown in Table 3.

Table 2 comparison of serum levels of 25(OH)D3, 1,25(OH)2D3,β CTX, P1NP in rats after 8, 12 16 weeks of modeling intervention(n=10, ±s)

Table 2 comparison of serum levels of 25(OH)D3, 1,25(OH)2D3,β CTX, P1NP in rats after 8, 12 16 weeks of modeling intervention(n=10, ±s)

Note:Compared with blank control group, P＜0.01; Compared with model group, ●P＜0.01; Compared with western medicine control group, ★P＜0.05

IndexTimeblank control group model group Western medicine control group Traditional Chinese medicine experimental group FP 25(OH)D3(ng/L)8 the weekend 502.89±24.48 429.37±21.11▲446.34±21.24●473.60±19.10●★22.29 ＜0.01 12 the weekend 496.37±24.00 423.74±20.68▲448.56±21.42●478.66±21.95●★21.28 ＜0.01 16 the weekend 498.34±25.32 414.13±20.33▲450.77±21.22●486.12±21.84●★29.06 ＜0.01 1,25(OH)2D3(ng/mL)8 the weekend 596.62±34.59 506.95±32.38▲545.64±35.64●586.40±32.33●★14.78 ＜0.01 12 the weekend 600.30±28.59 501.54±35.67▲552.90±38.78●586.64±33.42●★16.44 ＜0.01 16 the weekend 603.94±43.36 495.82±34.18▲556.81±40.10●589.68±37.88●★16.40 ＜0.01 P1NP(ng/mL)8 the weekend 8.12±0.99 4.83±1.13▲ 6.33±1.17● 7.65±0.90●★19.73 ＜0.01 12 the weekend 8.09±1.38 4.26±1.58▲ 6.22±1.05● 8.05±1.26●★18.74 ＜0.01 16 the weekend 7.94±1.07 3.38±1.06▲ 6.43±1.08● 7.99±1.34●★35.68 ＜0.01 8 the weekend 20.69±3.6630.25±2.56▲25.28±1.51●22.50±2.51●★24.32 ＜0.01 12 the weekend 21.65±3.4832.90±2.08▲25.47±2.22●21.20±3.66●★33.77 ＜0.01 16 the weekend 21.88±1.6832.57±2.42▲23.84±2.63●20.79±2.87●★48.03 ＜0.01 β-CTX(μg/L)

Table 3 Pearson correlation analysis of serum vitamin D3 ［25(OH)D3, 1,25(OH)2D3］ with bone turnover index (P1NP and β-CTX) and bone mineral density in rats with alcoholic osteoporosis

4. Discussion

Long-term high dose intake of alcohol can induce osteopenia through necrotic apoptosis of osteoblasts, thereby impairing bone remodeling, reducing bone mass, and increasing the risk of osteoporosis and fracture [12].At present, adequate intake of calcium and vitamin D is the core program for prevention and treatment of osteoporosis [13]. Long-term and high-dose drinking can inhibit bone formation level of mature bone rats, disrupt the balance of bone turnover, and then lead to progressive bone loss and decreased bone mineral density [14]. Therefore, in this study, we established a rat model of alcoholic osteoporosis to observe the prevention and treatment effects of Shengsui Jiangu capsule on alcoholic osteoporosis and related mechanisms. The experimental results showed that Shengsui Jiangu capsule can effectively improve bone mineral density and regulate bone conversion level of AOP rats,and has a significant effect on prevention and treatment of alcoholic osteoporosis.

Bone turnover, as an important process to maintain bone injury repair and mineral stability and balance in vivo, is affected by many factors. As a fat-soluble vitamin D active steroid molecules of vitamin, its main function is to maintain the serum calcium and phosphorus metabolism of bone mineral deposit stability,by monitoring the vitamin D3serum index in the body and can assess vitamin nutritional status, so as to achieve early found that changes in bone, and the purpose of prevention and treatment of osteoporosis in time [15]. As the main storage mode of vitamin D3in the body,25(OH)D3has a longer half-life than 1,25(OH)2D3, which can effectively maintain the dynamic stability of bone conversion and serum calcium level. The metabolizing active ingredient,1,25(OH)2D3, can enhance the ability of small intestinal mucosal cells to synthesize calcanin, improve the ability of kidney to reabsorb serum calcium and phosphorus, promote the deposition of bone mineral salts, improve bone mineral density, and improve the expression of bone formation markers. In addition, deficiency of 1,25(OH)2D3can reduce serum calcium content and increase osteoclast activity, stimulate accelerated dissolution of bone,maintain stable serum calcium level, and enhance the expression of bone resorption markers. P1NP is a metabolic product of type 1 collagen, which accounts for more than 90% of organic bone in the body, and is stored in osteoid by protease, which can more sensitize to reflect the state of bone formation in the whole body [7]. β-CTX,as a type 1 collagen crosslinking stabilizing collagen structure, can be released with the degradation of type 1 collagen in the process of bone resorption, which can sensitively and specifically reflect bone resorption [8].

According to the symptoms of alcoholic osteoporosis in Chinese medicine, alcoholic osteoporosis is classified as "bone impotence"and "bone blight" [16]. The main etiology and pathogenesis of alcoholic osteoporosis is mainly deficiency of liver, spleen and kidney, and alcohol addiction, which ultimately damages muscles and bone marrow [17]. From the "medical science" cut from the Shengsui Jiangu capsule, according to the characteristics of the alcoholic etiology and pathogenesis of osteoporosis, select your medicine tortoise plastron and antler glue, invigorating the bones,filling pulp, nourishing congenital, studies have shown that [18-19],antler glue and tortoise plastron effective ingredients can effectively improve the serum calcium phosphorus metabolism, regulating the osteogenetic differentiation and bone transform. Raw huangqi,Prepared rehmannia root, Atractylodes, etc as an official medicine,not only can dry wet water, hangover cure poison phlegmy wet, can nourish the day after tomorrow, the pharmacological studies [20-21]suggest that astragalus membranaceus and prepared rehmannia root,can be directly or indirectly affect and regulate bone microstructure,inhibiting osteoclast differentiation, and has a role in regulating on the cardiovascular system. Pueraria root, Salvia miltiorrhiza,Rhizoma drynariae, Euloides ulmoides and other drugs are used to cure alcoholism, promote blood stasis and relieve pain. Pueraria root not only has the effect of antialcoholism, but its extract puerarin has been proved to regulate oxidative stress state, maintain normal bone conversion efficiency and improve bone mineral density [22].Tanshinone ⅱ A, the active ingredient of Salvia miltiorrhiza, and Rhizoma drynariae can regulate the process of bone conversion and improve bone metabolism [23]. Eucommia ulmoides can promote osteoblast differentiation and increase bone mineral density by stimulating antioxidant stress proteins [24].

Modern medicine believes that alcohol can not only directly or indirectly poison bone cells and bone tissues and inhibit the proliferation of fibroblasts in bone marrow, but also indirectly destroy the dynamic balance between bone formation and bone absorption by affecting liver and kidney functions and organic molecules in serum, leading to bone reduction [25]. Long-term alcohol intake can reduce the activity of 1 hydroxylase in kidney, leading to the decrease of serum vitamin D3index (25(OH)D3and 1,25(OH)2D3),slowing down serum calcium and phosphorus metabolism and bone matrix calcification [26]. Previous studies of this project have shown that Shengsui Jiangu capsule has the following effects on rats with alcoholic osteoporosis :(1) It significantly improves biomechanical indexes of rat femur and increases bone strength and toughness [27].(2) Improve bone mineral density, maintain the dynamic balance of bone mineral production and loss, and improve bone metabolism disorders [28]. (3) The activation of Wnt signaling pathway interferes with the expression of lipid transcription gene PPAR γ 2 to achieve the prevention and treatment of alcoholic osteoporosis [17]. (4)Increase serum calcium, phosphorus and alkaline phosphatase levels,improve bone metabolism, reduce bone breakage rate, and inhibit bone loss [29]. (5) It can affect the expression of TNF-α and TGF-β1 in bone tissue, reduce the rate of empty bone lacunae, maintain the balance of bone metabolism, and repair part of osteonecrosis [30].This study found that Shengsui Jiangu capsule could significantly improve bone mineral density in AOP rats, and its mechanism may be closely related to promoting serum calcium absorption, regulating bone conversion level and improving bone microstructure of AOP rats.

In conclusion, Shengsui Jiangu capsule can promote the absorption of calcium ions in serum, maintain the stability and balance of bone repair and minerals during bone conversion, and improve bone density. From the perspective of vitamin D3absorption and bone conversion levels, this study explained the changes of bone mineral density in rats with alcoholic osteoporosis and the effect and mechanism of Shengsui Jiangu capsule on alcoholic osteoporosis,providing ideas and reference for the clinical application and research of Shengsui Jiangu capsule in the future.

Author's contribution:

Ren Shujun: Obtained funds and coordinated experiments; Gao Wei and Yu Changjiang. Literature retrieval and paper writing. Liang Yanlin, Tan Fuzhu: Recording and statistical experimental data; Li Yuanfeng, Xu Weiming, Xu Xilin: Daily maintenance of laboratory and update of consumables.

Conflict of interest:

No authors have any conflict of interest.