Effect of Shengsui Jiangu capsule on TNF-α, TGF-β1, and empty bone lacuna in rats with alcoholic osteoporosis

Shu-Jun Ren, Jun-Tong Liu, Chang-Jiang Yu, Lei Jiang, Fu-Zhu Tan, Yi-Wei Shen, Xi-Lin Xu?

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. Affiliated Chinese Medicine Hospital, Chongqing Three Gorges Medical College, Chongqing 404000, China

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

Keywords:Alcoholic Osteoporosis Shengsui Jiangu capsule TNF-α TEGmFp-t yβ b1

ABSTRACT Objective: To investigate the effect of postgraduate Shengsui Jiangu Capsule on tumor necrosis factors TNF-α,transforming growth factor TGF-β1 and empty bone lacuna in rats with alcoholic osteoporosis. Method: 120 SD rats were randomly divided into 4 groups, 30 rats in each group: blank group, model group, western medicine control group and traditional Chinese medicine intervention group.Liquor gavage was used to model the disease, except for the blank group. The western medicine control group was gavaged with a suspension of calcium carbonate and afad3, while the Chinese medicine intervention group was gavaged with a suspension of Shengsui Jiangu capsules combined with 0.9% saline solution injection.The values of TNF-α and TGF-β1 in femora were measured after 8, 12, and 16 weeks of intervention.After dissecting out the femoral head using the HE( hematoxylin-eosin staining)staining method, the bone lacunae were observed under a light microscope, and the empty bone lacunae rate was calculated for intergroup comparison. Results: Compared with the blank group, the model group had a significantly higher percentage of TNF-α and empty bone lacunae, and a significantly lower percentage of TGF-β1 (P ＜ 0.01).Compared with the model group, the rates of TNF-α and empty bone lacunae in the western medicine control group and the Chinese medicine intervention group were significantly decreased, and TGFβ1 was significantly increased (P ＜ 0.01).The traditional Chinese medicine intervention group was more effective than the western medicine control group (P ＜ 0.05). Conclusion: Shengsui Jiangu capsule attenuates the expression of TNF-α, enhances the expression of TGF-β1,decreases the rate of empty bone lacunae, and achieves a protective effect against alcoholic osteoporosis in rats with alcoholic osteoporosis.

1. Introduction

Alcohol-Induced Osteoporosis[1](AOP).It refers to the whole body bone disease in which the bone structure degenerates after long-term intake of too much alcohol, resulting in reduced bone mass and prone to fracture.According to the epidemiological survey results, the per capita annual consumption of pure alcohol is as high as 3.6L[2].Studies have shown that alcohol can easily lead to bone metabolic imbalance, affect the distribution of bone trabeculae, increase the risk of fracture, and even cause alcoholic bone disease.Therefore, it is more important to improve the relevant research for the prevention of alcoholic osteoporosis[3].TNF-α and TGF-β1 regulates the formation of bone, mainly including cell differentiation, proliferation and functional activities [4],which is closely related to the metabolic process of bone.TNF-α it is a factor that can affect the metabolic balance of bone and has extensive activity. It is one of the strongest active factors found so far[5];TGFβ1 has the function of active factor[6-8] regulating cell biochemistry and growth.Empty bone lacunae can directly reflect the degree of bone destruction under the microscope. Whether the rate of empty bone lacunae is reduced is an important index to detect and repair bone destruction[9].In this experiment, the rat model of alcoholic osteoporosis was established to study the effect of Shengsui Jiangu Capsule on in alcoholic osteoporosis rats TNF-α,TGF-β1 and empty bone lacuna rate, so as to provide a theoretical basis for guiding clinical treatment.

2. Materials and methods

2.1 Experimental animals

120 adult male SD rats (clean grade) with an average age of 6 months were supplied by the experimental center of Heilongjiang University Of Traditional Chinese Medicine,weight220±20g,Certific ate Number:SCXK(hei)20160103.

2.2 Experimental drugs and equipment

Shengsui Jiangu Capsule(Preparation room of Heilongjiang First Hospital of traditional Chinese Medicine,the numberZ20110036.The Executive standard:HEIZ-ZJ-0560-2010.Specifications:0.35g/grain);Calcium Carbonate Tablets(Specifications:500mg/slice,Jilin Wantong Pharmaceutical Co., Ltd.Approval number:Guoyao Zhun ZiH10980201);Alpha D3((Teva Pharmaceutical Industries Ltd.Approval number:Guoyao Zhun ZiJ20130162);Immunohistochemi cal Kit:SP Kit Box.(Mouse Substance P receptor SP-RELISA Kit)(Shanghai Xinyu Biotechnology Co., Ltd.Approval number:SP-0023);White spirit(Specifications:500ml/bottle,Hongxing 55°strong spirits usu,Beijing Hongxing Co., Ltd.Approval number:B1707039.Executive standard:QS1100-1501-0349);0.9%NaCl injection(Specifications:500ml/bag,Sichuan Pacific Pharmaceutical Co., Ltd.Approval number:1706150505);Hematoxylin staining solution and eosin staining solution(Shanghai Kehui Biotechnology Co., Ltd);Electron microscope(XK-DS500A,Shenzhen xinico photoelectric Co., Ltd).

2.3 Experimental method

2.3.1 Experimental model groupingAfter 1 week of adaptive feeding in the experimental animal center of Heilongjiang University Of Traditional Chinese Medicine, the rats were randomly divided into 4 groups with 30 rats in each group.

2.3.2 Establishment of experimental animal model

Model Group, Western Medicine Control Group And Traditional Chinese Medicine Intervention Group: Forenoon,white spirit was used for gastric lavage.The dose is 10ml/kg(Alcohol quantity 4.4g/10mL).Model Group: In the afternoon, the same amount of 0.9% NaCl injection was given by gavage. Once a day.

Western Medicine Control Group: In the afternoon, calcium carbonate and alpha D3 suspension and 0.9%NaCl injection preparation suspension were used to gavage(The dosage standard of calcium carbonate is 85 mg/l0ml/kg,the dosage standard of alpha D3 is 0.05μg/10mL/kg).Once a day.

Traditional Chinese Medicine intervention group: In the afternoon,Shengsui Jiangu Capsule and 0.9% NaCl injection were used to prepare suspension for intragastric administration (The dosage standard was 4.7g/10ml/kg).

Blank group: The same amount of 0.9% NaCl injection was given by gavage twice a day in the morning and afternoon.The intragastric volume of rats in each group was 10ml/kg and the intragastric administration time was 16 weeks.During the experiment, rats were fed under the same condition, and the dosage was adjusted according to weekly body weight[10].

2.4 Sampling and testing methods

At 8, 12 and 16 weeks after the intervention, 10 rats were randomly selected from each group.In the morning, the right upper femur was taken for immunohistochemical detection after being killed by spinal dislocation on an empty stomach (taking more than 10 hours on an empty stomach as the standard).The extracted femoral tissue was made into a slide specimen, and the reagent was detected under the microscope. The enzyme labeled antibody was used to react with the tissue.Labeled TNF-α、TGF-β1 cytokines.Five visual fields were randomly selected to record TNF-α,TGF-β1 value under electron microscope.After the last gavage, the rats were kept in an fasting state, and samples were collected 12 hours later for detection[11].

Dissect and free the left femur,it was fixed in 4% paraformaldehyde solution for 24 hours.After that, it was placed in 15% EDTA(ethylenediamine tetraacetic acid) to fully decalcify.Conventional dehydration, paraffin embedding, section thickness 5μm,HE staining was performed.The changes of bone trabeculae, bone marrow cavity, subchondral bone cells and backbone tissues and empty bone lacunae were observed under light microscope.10 high-power visual fields were randomly selected in each group, 100 bone lacunae were counted in each visual field, the number of empty bone lacunae was counted, the percentage of empty bone lacunae was calculated, and micrography was performed.

2.5 Data analysis

All data were processed by spss21.0 software, and the measurement data were expressed in±s. The comparison between each group was analyzed by one-way ANOVA, and the difference was statistically significant (P ＜ 0.05).

3. Results

3.1 8,12,16 weekend in each group of rats femoral tissue TNF-α and TGF-β1 comparison of positive cell number

According to the test results at the end of 8, 12 and 16 weeks of modeling intervention, compared with the blank group,TNF-α in the model group and empty bone lacuna rate increased significantly,TGF-β1 decreased significantly (P ＜ 0.01).It is suggested that a large amount of alcohol intake can lead to bone destruction and disorder of bone metabolic balance in experimental rats.Compared with model group, in western medicine control group and traditional Chinese medicine intervention group TNF-α And empty bone lacuna rate decreased significantly,TGF-β1 increased significantly (P ＜ 0.01).It is suggested that the two ways of drug intervention can maintain the balance of bone metabolism and alleviate the bone damage in experimental rats.Compared with the western medicine control group, the improvement of indexes in the traditional Chinese medicine intervention group was better than that in the western medicine control group (P ＜ 0.05).

Seeing table 1,table 2 and table 3.

3.2 HE staining in bone tissue of rats in each group(×400)

According to the observation results of HE staining light microscope of bone tissue of experimental rats at the end of 8th,12th and 16th weeks:the bone trabeculae in the blank group were arranged orderly without fracture.The shape of medullary cavity is regular without enlargement, and there are a large number of red bone marrow.The number of bone lacunae over trabecular bone is rare.The surface of subchondral bone is smooth.In the model group,the number of trabeculae decreased, became thinner and arranged disorderly.Medullary cavity expands, red bone marrow decreases,and yellow bone marrow (adipose tissue) increases.The number of bone lacunae over trabeculae and shaft was higher than that in blank group.The bone cells in the backbone are arranged irregularly, and a few nuclei are pyknosis.In the western medicine control group, the bone trabeculae were arranged irregularly without obvious fracture.The morphology of medullary cavity was irregular, the red bone marrow increased and the yellow bone marrow decreased compared with the model group.Compared with the model group, the number of bone lacunae over trabecular bone and shaft decreased.The bone trabeculae in the traditional Chinese medicine intervention group were arranged regularly without fracture.The morphology ofmedullary cavity was not significantly enlarged, and there were a large number of red bone marrow.The number of bone lacunae over trabeculae and shaft was less than that in model group.The surface of subchondral bone is more smooth.The osteocytes at all levels under the cartilage are arranged orderly and evenly, and the mature osteocytes are relatively increased.The bone cells of the shaft are arranged neatly with a small amount of nuclear pyknosis. Seeing table1.

Table 1 8,12,16 weekend in each group of rats femoral tissue TNF-α comparison of positive cell number (±s)

Table 1 8,12,16 weekend in each group of rats femoral tissue TNF-α comparison of positive cell number (±s)

Note: compared with the blank group, *P ＜ 0.01; Compared with the model group, #P＜ 0.05, aP ＜ 0.01; Compared with western medicine control group, bP ＜ 0.05.

Groupthe end of 8th Weeksthe end of 12th Weeksthe end of 16th Weeks Blank group31.71±4.9731.98±5.0331.66±5.06 Model Group51.33±5.07*57.47±4.34*63.12±5.47*Western Medicine Control Group50.16±4.91#48.93±5.14#47.47±4.83#TCM Intervention Group39.76±4.77ab38.27±5.34ab36.13±5.26ab F Value F=35.476F=51.654F=73.835 P ValueP＜0.01P＜0.01P＜0.01

Table 2 8,12,16 weekend in each group of rats femoral tissue TGF-β1 comparison of positive cell number(±s)

Table 2 8,12,16 weekend in each group of rats femoral tissue TGF-β1 comparison of positive cell number(±s)

Note: compared with the blank group, *P ＜ 0.01; Compared with the model group, #P＜ 0.05, aP ＜ 0.01; Compared with western medicine control group, bP ＜ 0.05.

Groupthe end of 8th Weeksthe end of 12th Weeksthe end of 16th Weeks Blank group60.09±3.3760.17±3.4160.22±3.34 Model Group51.63±5.13*44.17±4.63*37.88±4.17*Western Medicine Control Group47.87±5.13#49.13±4.38#51.11±4.46#TCM Intervention Group57.19±5.67ab56.97±4.25ab58.53±5.25ab F ValueF=12.017F=30.301F=54.476 P ValueP＜0.01P＜0.01P＜0.01

Table 3 8,12,16 weekend in each group of rats femoral tissue comparison of Lacuna rate of empty bone(±s)

Table 3 8,12,16 weekend in each group of rats femoral tissue comparison of Lacuna rate of empty bone(±s)

Note: compared with the blank group, *P ＜ 0.01; Compared with the model group, #P＜ 0.05, aP ＜ 0.01; Compared with western medicine control group, bP ＜ 0.05.

the end of 16th Weeks Groupthe end of 8th Weeksthe end of 12th Weeks Blank group13.00±1.6313.20±1.6212.90±1.66 Model Group20.80±1.75*22.10±2.08*23.00±1.49*Western Medicine Control Group19.70±1.64#19.20±1.55#18.10±1.91#TCM Intervention Group18.60±1.43ab17.90±1.79ab16.20±1.55ab F ValueF=136.307F=137.389F=133.282 P ValueP＜0.01P＜0.01P＜0.01

Figure 1 The results of HE staining in bone tissue of rats in each group(×400)

4. Discussion

Long term excessive alcohol intake can cause thinning of bone cortex, reduction of bone trabeculae, reduction of bone mass and changes of bone metabolism, resulting in increasing risk of fracture.The dynamic balance between osteoblasts and osteoclasts is the basis for maintaining the balance of bone metabolism[12].The effect of osteoblasts on bone growth is less than that of osteoclasts on bone resorption, which is the root cause of osteoporosis[13].Research shows that TNF-α in the early stage of osteoclasts, it can stimulate and form new osteoclasts through the conduction of cell surface receptors, and indirectly promote the rapid differentiation of osteoclasts and the proliferation and functional activity of osteoclasts.So TNF-α can inhibit the withering of osteoclasts and lead to the increase of bone resorption[14].Meanwhile, TNF-α can inhibit the differentiation of pre osteoblasts and inhibit the process of new bone formation and calcification, so as to reduce the number of bone trabeculae and bone cortical density[15].The differentiation of osteoblasts is the main source of bone matrix, while TGF-β1 is one of the most abundant active factors in bone matrix.So TGF-β1 can promote the formation of new bone and accelerate the process of new bone calcification by stimulating osteoblasts[16].Simultaneous TGF-β1 can promote the synthesis and recombination of active factors in pre osteoblasts, promoting the formation of osteoblasts and enhance their functional activity, and reduce their proliferation and activity by inhibiting the expression of osteoclasts[17].TGFβ1 promote the proliferation of osteoblasts and promote their transformation to mature type, so as to reduce bone resorption and maintain the dynamic balance of bone metabolism.Therefore,TGFβ1 plays an important role in bone repair and construction.So TGFβ1 is considered to be a factor closely related to the balance of bone metabolism[18].

According to the clinical symptoms of alcoholic osteoporosis,traditional Chinese medical science classifies it as “bone flaccidity”.Traditional Chinese medicine believes that deficiency of kidney essence and deficiency of kidney qi are the root causes of bone changes[19].Therefore, the pathological changes of alcoholic osteoporosis are in bone but the root is in kidney[20].The spleen is the acquired foundation, the main muscle and the biochemical source of Qi and blood.Kidney deficiency leads to spleen metaplasia and weakness, and spleen deficiency leads to biochemical passivity to nourish the kidney[21].Therefore, tonifying the kidney and spleen is the main treatment principle for alcoholic osteoporosis.Shengsui Jiangu Capsule is made from Antler capsules in YiXueZhengZhuan.It is mainly made of tortoise shell and antler glue. Antler glue is good at tonifying kidney and Yang, and tortoise shell is good at tonifying qi and Yin.When the two drugs are used together,yin and yang are replenished together. Yang gets Yin to help and biochemistry is infinite. Yin gets yang to rise and the spring source is inexhaustible. Yin and Yang help each other, so it can replenish the kidney to fund congenital diseases.Taking Astragalus membranaceus and Rehmannia glutinosa as official medicines, Astragalus membranaceus is good at raising Yang and replenishing qi,Rehmannia glutinosa is good at replenishing blood and nourishing Yin, and blood is the mother of Qi.Qi is the beauty of blood, and Yin and yang are supplemented at the same time, so it can strengthen the spleen and supplement,and the spleen is transformed into infinity[22].Previous studies have shown that Shengsui Jiangu Capsule has a good therapeutic effect on alcoholic osteoporosis[23].

Literature shows that excessive drinking can cause endocrine disorders and promote TNF-α expression of.Thus accelerating the formation of osteoclasts[24].Shengsui Jiangu Capsule has the effects of tonifying the kidney and spleen and strengthening muscles and bones. It can regulate endocrine disorders and promote the formation of osteoblasts.With the correction of endocrine disorder, TNFαwas inhibited expression of.Shengsui Jiangu Capsule regulates the metabolism in rats, ensures the normal Wnt / BMP-2 pathway and promotes the secretion of TGF-β1 by bone cells.At the same time, it inhibits the apoptosis of osteoblasts and promotes the secretion of TGF-β1 by osteoblasts, and can make IGF-1 growth promoting factor self phosphorylation, so as to promote TGF-β1 expression.Hip joint is the main weight-bearing joint. Under normal circumstances, the bone trabeculae in the femoral head are closely arranged.In the compression stress and shear stress concentration area of femur, such as femoral calcar, the bone trabeculae increased significantly, which is the result of long-term evolution.However,when a pathogenic factor breaks the balance of bone metabolism,osteoclast activity increases, osteoblast activity decreases, new bone formation decreases and osteocyte necrosis increases.Then the number of empty bone lacunae increased, that is, the rate of empty bone lacunae increased[25], resulting in osteoporosis.The rate of empty bone lacuna in normal femoral head is less than 13%. Empty bone lacuna is the direct evidence of bone cell death, which directly reflects the degree of bone necrosis[26].Shengsui Jiangu Capsule can reduce the rate of empty bone lacunae by adjusting bone metabolism,suggesting that it has a certain repairing effect on bone damage in rats with alcoholic osteoporosis.

The experimental results show that Shengsui Jiangu Capsule can reduce TNF-α in bone tissue of rats with alcoholic osteoporosis Enhance the expression of TGF-β1 expression.It can reduce the rate of empty bone lacunae, maintain the dynamic balance of bone metabolism and repair bone necrosis, so as to prevent and treat alcoholic osteoporosis and provide basis and reference for clinical treatment.