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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 5  |  Issue : 4  |  Page : 243-249

Effect of total flavonoids of Herba Taxilli on osteoporotic rats induced by retinoic acid


School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, Henan Province, China

Date of Submission10-Dec-2018
Date of Decision03-Apr-2019
Date of Acceptance25-Apr-2019
Date of Web Publication03-Dec-2019

Correspondence Address:
Prof. Ying Cui
Henan University of Chinese Medicine, Zhengzhou 450046, Henan Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_23_19

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  Abstract 


Objective: The objective was to study the effect of total flavonoids of Herba Taxilli (TFHT) on osteoporotic (OP) rats induced by retinoic acid (RA) and explore its mechanism. Methods: Three-month-old female Sprague Dawley rats were randomly divided into six groups, each with ten rats, i.e. normal control group, model group, and positive control group and three TFHT groups (at high-, middle-, and low-doses). Except the normal control group, rats of all the other groups were administered RA by gavage (70 mg/kg/day) every morning for 14 days to establish an OP model; at the same time, all the rats were given Corresponding therapeutic drug by gavage for 28 days: rats in the three TFHT groups were given TFHT (400, 200, and 100 mg/kg/day, respectively) and rats in the positive control group were given Long mu Zhuang gu Granule 300 mg/kg/day. Rats in the model group and normal group were given an equal volume of 1% sodium carboxymethyl cellulose sodium suspension. After the experiment, blood was taken to measure the levels of serum calcium (Ca2+), alkaline phosphatase (ALP), and tartrate-resistant acid phosphatase (TRAP); the spleen, uterus, and ovary were dissected to determine the organ coefficient; the left hind limb femur was dissected to determine bone mineral density (BMD), bone dry weight (BDW), bone wet weight (BWW), bone diameter (BD), bone length (BL), bone dry weight/rat weight (BDW/W), bone wet weight/rat weight (BWW/W), bone dry weight/bone wet weight (BDW/BWW), bone dry weight/bone length (BDW/L), and bone wet weight/bone diameter (BWW/D); and the right hind limb femur was dissected to observe the bone path morphological changes. Results: Compared with the model group, the serum Ca2+ content, uterus plus ovary coefficient, BMD, BDW, BDW/W, BWW/W, BDW/BWW, BDW/L, and BWW/D in the TFHT groups increased, whereas the spleen coefficient, ALP, and TRAP decreased in the TFHT groups, and the differences were statistically significant (P < 0.05 orP < 0.01). Conclusion: TFHT was found effective in treating OP in rats induced by RA; the main mechanisms were increasing the serum Ca2+ content and reducing the level of ALP and TRAP.

Keywords: Osteoporosis, retinoic acid, total flavonoids of Herba Taxilli


How to cite this article:
Li YY, Cui Y, Wang HL, Feng J, Li LL. Effect of total flavonoids of Herba Taxilli on osteoporotic rats induced by retinoic acid. World J Tradit Chin Med 2019;5:243-9

How to cite this URL:
Li YY, Cui Y, Wang HL, Feng J, Li LL. Effect of total flavonoids of Herba Taxilli on osteoporotic rats induced by retinoic acid. World J Tradit Chin Med [serial online] 2019 [cited 2019 Dec 13];5:243-9. Available from: http://www.wjtcm.net/text.asp?2019/5/4/243/271963




  Introduction Top


Osteoporosis (OP) is characterized by a decrease in bone mass as well as a deterioration of the bony architecture, resulting in an increased risk of fracture. Patients mainly suffer from deformed vertebrae, fracture, and respiratory system disorder.[1],[2]

In general, elderly women and menopausal women more likely suffer from OP. China has the largest population in the world; with improvement in living standard and increasing life span, the aging population is growing rapidly.

Therefore, it is important to prevent and cure OP in the elderly. While until now, there is no ideal drug to completely cure OP. Estrogen replacement therapy for OP is commonly used in Western medicine; it plays an effective role in alleviating bone loss in postmenopausal women. The drugs commonly used are diethylstilbestrol, estradiol, etc., yet they are potential carcinogens.[3],[4]

Herba Taxilli, which was first recorded in the Shen Hong's herbal classic as an upper product, is from the dry stem branches and leaves of Taxillus chinensis (DC.) Danser, Loranthaceae family. It is bland in nature, bitter and sweet in flavor, and attributive to the liver and kidney meridians. Its actions are expelling wind and dampness, invigorating the liver and kidney, strengthening tendons and bones, and preventing miscarriage. Clinically, it is mainly used for rheumatic arthralgia with weakness of waist, knees, and muscles; uterine bleeding; excessive fetal movement, gravida vaginal bleeding,[5] etc. Recent studies have shown that the main chemical components of Herba Taxilli are flavonoids, such as quercetin, quercitrin, acutin, rutin, and a small amount of dextral catechol.[6] Flavonoids mainly found in plants have various pharmacological activities, which are a hot topic in the research of new drugs against OP. Herba Taxilli possesses antitumor, antioxidative, anti-allergic, anti-inflammatory, analgesic, antihypertensive, hypolipidemic, memory-enhancing, and nerve-protecting effects. It is mainly used for the treatment of OP, cardio-cerebro-vascular diseases, gynecologic diseases,[7] etc.

In an earlier study,[8] we found that Herba Taxilli decoction has some therapeutic effect on OP in rats induced with retinoic acid (RA). Other studies have shown that Herba Taxilli has the ability of regulating bone metabolism, inhibiting excessive bone absorption, increasing the activity of bone cells, making bone formation exceed bone absorption, and improving OP.[9]Herba Taxilli can treat OP by promoting the expression of osteoprotegerin and decreasing the level of interleukin-1.[10] Therefore, an experiment on the effect of total flavonoids of Herba Taxilli (TFHT) on OP in rats was carried out.


  Experiment Materials Top


Experiment animals

Female Sprague-Dawley (SD) rats (specific pathogen free, about 230 g) were purchased from the Laboratory Animal Center of Henan Province, license no. SCXK (YU) 2015-0004, batch no. 41003100004021. These rats were housed in individual ventilation cages (IVCs) in the animal room of Henan University of Chinese Medicine (HUCM), fed with standard feed the temperature was controlled at 20°C, and the breeder was responsible for feeding and management. The feed was purchased from Henan Experimental Animal Center, license no. SCXK (YU) 2015-0005, batch no. 41000100003218.

Extraction of total flavonoids of Herba Taxilli

Herba Taxilli cultivated in Anhui was purchased from Zhang Zhongjing Pharmacy in Henan province; its production date was March 24, 2016. Professor Chen Sui-Qing of HUCM identified it from the dry stem branches and leaves of T. chinensis (DC.) Danser, Loranthaceae family. Herba Taxilli was used as the raw material to obtain TFHT, via condensing reflux extraction with 70% ethanol (twenty times weight, twice, 1.5 h/time), vacuum concentration, AB-8 macro reticular adsorbent, and then elution with water and 50% ethanol. Finally, the TFHT was grinded into reddish brown powder after vacuum concentration and low-temperature drying. The content of flavonoids in the extracts determined by Al (NO3)3 colorimetry was >56%. For the experiment, required concentration suspensions were made by adding a certain amount of the powder in 1% sodium carboxyl methyl cellulose (sodium carboxyl methyl cellulose, CMC-Na), dissolving with ultrasonic equipment.

Drugs and reagents

RA (batch no. 2322B92, America Amresco Inc.), Long mu Zhuang gu Granule (LZG) (batch no. 160761, Wuhan Jianmin Pharmaceutical Group Co., Ltd), CMC-Na (batch no. 20160728, Tianjin Guang-fu Fine Chemical Institute), alkaline phosphatase (ALP) kits (batch no. 20170110, Nanjing Jiangcheng Bioengineering Institute); serum calcium (Ca2+) kits (batch no. 20170110, Nanjing Jiangcheng Bioengineering Institute), and TRACP kits (batch no. 20170411, Nanjing Jiangcheng Bioengineering Institute) were used in the study.

Experimental instruments

IVC for rats (model: IVC-II, Suzhou Fengshi Experimental Animal Equipment Co. Ltd), high- speed freezing centrifuge (model: TGL-16gR, Shanghai Anting Scientific Instrument Factory), vacuum drying oven (model: DZF-6020, Shanghai Precision Experimental Equipment Co., Ltd.), electric heat dryness box (model: GZX-9070, Shanghai Bo Xun Industrial Co., Ltd.), electronic balance (model: Acculab, Sartorius Scientific Instrument Co., Ltd.), dual-energy X-ray absorptiometry (DXA) (model: Discovery, Hologic, Inc), and microplate reader (model: BioTek Instruments, Inc.) were used in the study.


  Experimental Methods Top


Rat model establishment and administration

After 5-day adaptation with access to free drinking and eating, sixty 3-month-old female rats were randomly divided into six groups (n = 10 in each): normal group, model group, positive control group and three TFHT groups (at high, middle, and low doses). Except normal group, rats in all groups were given RA (suspended in 1% CMC-Na; 70 mg/kg/day, ig) once in the morning for 14 days; meanwhile, the corresponding drugs or distilled water was given once in the afternoon for 28 days. Rats in the TFHT groups were given TFHT (400, 200, and 100 mg/kg/day, respectively, ig), and rats in the positive control group were given LZG (300 mg/kg/day, ig). Rats in the normal and model groups were given distilled water (100 mL/kg·d−1, ig). The dosage of the mulberry parasitic decoction was selected by Wang Jing based on the “Study on the mulberry parasitic substance and the classics based on the “sickness syndrome-effect-biological sample analysis” method.” Moreover, the extraction rate of total flavonoids from mulberry parasitic was 5.5%. According to the efficacy results of pre-experiment, it was finally determined that the low-dose group was 100 mg/kg, the middle-dose group was 200 mg/kg, and the high-dose group was 400 mg/kg. In the experimental results, the problem of dose independence is mainly because there may be a slight error in the measurement of each indicator and individual differences in each animal may also affect; hence, consistency cannot be achieved well. In the experiment, every rat was weighed every 4 days with electronic scales, and the results (g) were recorded. After the last administration, rats in each group were fasted 24 h. On the 29th day, after the rats were anesthetized, blood from the abdominal aorta, spleens, uteruses, ovaries, left hind limb and right hind limb were taken were taken. Then, serum was isolated by high-speed freezing centrifuge at 8000 rpm for 10 min from blood and preserved in the refrigerator at −20°C.

Main outcome measures

  1. The levels of serum Ca2+, ALP, and tartrate-resistant acid phosphatase (TRAP) were measured following kit instructions
  2. Organ coefficient: The spleens, uteruses, and ovaries of rats were weighed with 1/10,000 electron balances, and the respective organ coefficients were calculated according to the following formula: Organ coefficient = visceral weight (mg)/body weight (g)
  3. The ratio of bone wet weight (BWW)/W, bone dry weight (BDW)/W, BDW/BWW, BWW/bone diameter (BD), and BWW/bone length (BL): The left hind limb femurs of rats in each group were measured for BWW, BDW, BD, and BL. Then, the ratios of BWW/W, BDW/W, BDW/BWW, BWW/BD, and BWW/BL were calculated
  4. Bone mineral density (BMD): BMD of the left femur in rats was measured by DXA
  5. Changes of bone path morphology: The right hind limb femur of rats was fixed with 10% formalin. After a week, the bone was decalcified and dehydrated, embedded in paraffin, and sliced into 5 μm successive coronal slices with a microtome. The slices were dried with a water bath and a slide drier, and then stained with hematoxylin and eosin (H and E) to evaluate the path morphology changes of caput femoris with optical microscopy.


Statistical analysis

The data were processed by SPSS 20.0 (SPSS Inc., Chicago, IL, USA) for Windows. Measurement data were expressed as mean ± SD (x̄s), and group comparison was performed using analysis of variance. P < 0.05 and P < 0.01 were considered statistically significant.


  Results Top


General observation

In the experiment, rats except in normal group, after given RA for a week, were found with different degrees of dry and sparse hair, bow back, reduced activity, reduced diet, weight loss, etc., especially significant in the model group. After RA was removed, the above-mentioned conditions in the LZG and TFHT groups were distinctively improved.

Effect of total flavonoids of Herba Taxilli on biochemical indexes in osteoporotic rats

Compared with normal group, in the model group, serum Ca2+ decreased significantly, and the level of ALP and TRAP increased statistically significantly (P < 0.01), which confirmed that the models were successfully made. Compared with the model group, in positive group, Ca2+ content increased statistically significantly (P < 0.01), and the level of ALP and TRAP decreased statistically significantly (P < 0.01); in high- and middle-dose TFHT groups, Ca2+ content statistically significantly increased (P < 0.05), and the level of ALP and TRAP statistically significantly decreased (P < 0.05 or P < 0.01). The results are summarized in [Table 1].
Table 1: Effects on biochemical indexes in osteoporotic rats (x̄ ±s, n=10)

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Effect of total flavonoids of Herba Taxilli on organ coefficient in osteoporotic rats

Compared with normal group, spleen coefficient in the model group increased statistically significantly (P < 0.01), and uterus plus ovary coefficient decreased statistically significantly (P < 0.05), which confirmed that the models were successfully made. Compared with model group, in positive and middle-dose THTF groups, spleen coefficient decreased statistically significantly (P < 0.01), and uterus plus ovary coefficient increased statistically significantly (P < 0.01). The results are shown in [Table 2].
Table 2: Effects on organ coefficient in osteoporotic rats (x̄ ±s, n=10)

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Effect of total flavonoids of Herba Taxilli on the bone mineral density and bone dry weight in osteoporotic rats

Compared with normal group, the BMD and BDW of left femur in the model group decreased statistically significantly (P < 0.05), indicating that the OP models were successfully made. Compared with model group, in positive control group and high- and middle-dose TFHT groups, the BMD of the left femur statistically significantly increased (P < 0.01) and the BDW increased statistically significantly (P < 0.05). The results are shown in [Table 3].
Table 3: Effects on the ratio of bone mineral density and bone dry weight in osteoporotic rats (x̄ ±s, n=10)

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Effect of total flavonoids of Herba Taxilli on the ratio of bone dry weight/W, bone wet weight/W, and bone dry weight/bone wet weight

Compared with normal group, in model group, the ratio of BDW/W and BWW/W statistically significantly reduced (P < 0.01) and BDW/BWW statistically significantly reduced (P < 0.05), which indicated that the models were successfully made. Compared with model group, in all the medication-administered groups, the ratio of BDW/W and BWW/W statistically significantly increased (P < 0.05) and BDW/BWW statistically significantly increased (P < 0.01). The results are shown in [Table 4].
Table 4: Effects on the ratio of bone dry weight/W, bone wet weight/W, and bone dry weight/bone wet weight in osteoporotic rats (x̄ ±s, n=10)

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Effect of total flavonoids of Herba Taxilli on the ratio of bone wet weight/L and bone wet weight/diameter in osteoporotic rats

Compared with normal group, the ratio of BWW/L and BWW/D in the model group statistically significantly reduced (P < 0.01), which confirmed that the models were successfully made. Compared with the model group, in the positive group, the ratio of BWW/L and BWW/D statistically significantly increased (P < 0.01), and in the three TFHT groups, the ratio of BWW/L and BWW/D statistically significantly increased (P < 0.05 or P < 0.01). The results are shown in [Table 5].
Table 5: Effects on the ratio of bone wet weight/L and bone wet weight/D in osteoporotic rats (x̄ ±s, n=10)

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Effects of total flavonoids of Herba Taxilli on path morphology changes of left femur in osteoporotic rats

By redid test, the difference of bone path morphology changes between normal group and model group was found statistically significant, which confirmed that the models were successfully made. Compared with the model group, bone path morphology changes in the three TFHT groups were statistically significant (P < 0.01), and in the positive group, they were statistically significant (P < 0.05), which showed that all the drugs could obviously improve the symptoms of OP. The results are shown in [Table 6].
Table 6: Effects on path morphology changes of left femur in osteoporotic rats (x̄ ±s,n=10)

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  Discussion Top


Understanding osteoporosis in traditional Chinese medicine

In the theory of traditional Chinese medicine (TCM), there is no OP, but according to the clinical manifestations and pathogenesis, this disease should be in the category of “bone atrophy,” “bone weakness,” “bone obstruction,” and so on [Figure 1]. In TCM, the kidney is considered as the origin of congenital constitution, which generates marrow and dominates bone. Essentials of Chinese Medical Classic says: The kidney stores essence, essence produces marrow, marrow generates bone, so the bone bases on kidney. The marrow is from the kidney essence, adequate kidney essence can make the bone marrow sufficient, bone marrow inside the bone and adequate bone marrow can strengthen bones. Deficiency of kidney essence causes insufficiency of bone marrow, resulting in loose bone, and then weakness, leading to “bone atrophy” and “bone weakness.” Therefore, the primary cause of OP is the damage of kidney essence; meanwhile, weakness of spleen and stomach, deficiency of liver blood, and extravasated blood are other important factors. Hence, kidney essence is closely related to bone metabolism. The deficiency of the kidney to a certain extent can lead to bone metabolism disorder, leading to OP.[11] Based on the efficacy of Herba Taxilli which can invigorate the liver and kidney and strengthen tendons and bones, it should be effective in treating OP.
Figure 1: Pathological slices of left femur in osteoporotic rats under optical microscope

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Understanding osteoporosis in modern medicine

OP is a common disease in middle-aged and elderly people. Its etiology and pathogenesis are complex. Modern medicine believes that OP is related to many factors, such as aging, hormonal level, heredity, cytokine level, and environment. According to clinical manifestation and pathogenesis, OP has two kinds: primary and secondary. Primary OP is divided into three types: idiopathic juvenile OP, postmenopausal OP, and senile OP, whereas secondary OP is mainly caused by diseases (such as endocrine and metabolic diseases, connective tissue diseases, kidney diseases, and digestive tract diseases) or due to drugs (such as anticancer drugs, immunosuppressants, and thyroxine).

Selection of model animals of osteoporosis

Rat is one of the most widely used animal models of OP. SD rats whose life span is usually 2–3 years have the advantages of rapid growth and short bone remodeling time. The epiphysis of male SD rats perhaps does not get arrest until the 30th month, whereas that of female rats get arrest at 6–9 months, then in metabolic stability at 10 months.[12] That is to say, the inactive stage of bone growth in female rats reaches earlier than that in male rats, and the growth period of physeal cartilage is shorter. Therefore, female rats were selected for the current experiment.

Selection of modeling method

In this study, RA was used to induce OP that is secondary to OP. RA derived from Vitamin A can promote osteoclast activity and bone absorption, and it does not decrease osteoblast activity. Bone formation is always less than bone absorption, resulting in OP.[13] The model of OP induced by RA in rats is widely used in OP drug development because RA is simple, stable, and reliable.[14]

Effects of total flavonoids of Herba Taxilli on the level of serum calcium, alkaline phosphatase, and tartrate-resistant acid phosphatase in model rats

The mode of modeling in each drug-administered group was the same as that in the model group, and several preexperiments were carried out in the early stage of the experiment. The model was explored and tested several times, and the modeling method was found to have good stability and high success rate.

Ca2+ is the core element of BMD and plays an important role, particularly in bone metabolism. Essentially bone metabolism is Ca2+ metabolism, that is, bone continues to release the stored Ca2+ into blood and then restores the Ca2+ from blood to bone, which ensures that the bone metabolism is steady.[15] Therefore, the increase of Ca2+ content in blood can help bone formation and calcification and inhibit Ca2+ in bone transfer to blood.[16] The results showed that the Ca2+ content in model group decreased significantly, indicating that Ca2+ was excessively lost and bone metabolism was abnormal after modeling. While in all the medication-administered groups, Ca2+ content statistically significantly increased (P < 0.05), suggesting that TFHT could make Ca2+ content tend to be normal, effectively prevent the Ca2+ metabolic disorder in OP rats, and maintain the metabolic balance of various inorganic elements. The level of ALP is usually used to evaluate bone turnover and formation and the activity of bone cells. When osteoporosis occurs, osteoblasts compensatory hyperactivity, osteoclast activity is enhanced, ALP release is increased, and bone minerals are reduced, inhibition of ALP is weakened, and ALP is also increased.[17] ALP, a specific enzyme produced by osteoclasts, has the effect of bone absorption by degradation of hydroxyphosphates in bone matrix.[18] It is a sensitive index to reflect osteoclast activity and bone resorption level.[19] The results showed that the levels of ALP and TRAP in model group increased statistically significantly (P < 0.01), indicating that RA increased the activity of osteoblasts and osteoclasts and accelerated the bone turnover. After the treatment of TFHT, the levels of ALP and TRAP decreased statistically significantly (P < 0.01), indicating that TFHT could inhibit the activity of osteoclasts, reduce bone absorption, increase the activity of osteoblasts, promote bone formation, decrease bone turnover, and increase bone mass.

Effect of total flavonoids of Herba Taxilli on bone mineral density in osteoporotic rats

BMD is an important indicator of bone strength, which mainly indicates bone mineral content, and is usually used as criteria for OP.[20],[21] The results showed that compared with normal group, in model group, the BMD of rats statistically significantly decreased (P < 0.01), the level of Ca2+ statistically significantly decreased (P < 0.01), the level of ALP and TRAP statistically significantly increased (P < 0.01), and BMD positively correlated with the level of Ca2+ and negatively correlated with the level of ALP and TRAP. That showed the decrease of BMD in the model group should due to increased bone metabolism, blood calcium transfer to bone calcium and the amount of bone resorption is greater than the amount of bone formation. Compared with the model group, the BMD of rats in each medication-administered group statistically significantly increased (P < 0.01), which showed that TFHT and LZG could effectively prevent the loss of BMD and reduce BMD in rats caused by RA. BDW and the ratio of BDW/W, BWW/W, BDW/BWW, BWW/D, and BWW/L from the left femur in rats can reflect its bone density in a sense.[22] The results showed that compared with the normal group, BDW and the ratio of BDW/W, BWW/W, BDW/BWW, BWW/D, and BWW/L in the model group decreased statistically significantly (P < 0.01), reflecting that RA could reduce the BMD of the left femur in rats. Although BMD is an important indicator of bone strength, the reconstruction of the trabecular bone also needs to consider other factors,[23] Therefore, in this study, the pathological changes of the femur were also detected to evaluate the degree of bone loss and the destruction of microframework.

Effect of total flavonoids of Herba Taxilli on organ coefficient in osteoporotic rats

Reproductive organs (uterus and ovary) in female rats are important organs to produce estrogen. Estrogen promotes bone formation and regulates bone metabolism. Therefore, the decrease of estrogen will increase the risk of OP.[24] Compared with the normal group, the uterine plus ovarian coefficient in the model group decreased significantly and spleen coefficient increased significantly, suggesting that RA could significantly damage the reproductive organs in rats and the spleen was in compensatory hypertrophy. Compared with the model group, in all the medication-administered groups, the uterine plus ovarian coefficient significantly increased and the spleen coefficient decreased statistically significantly (P < 0.01) and the compensatory hypertrophy of spleen also disappeared. This indicates that LZG and a certain dose of TFHT could prevent the damage to reproductive organs and the compensatory hypertrophy of spleen caused by RA in rats.

Effects of total flavonoids of Herba Taxilli on bone histomorphology of femur in model rats

At the end of the experiment, the left hind limb femur of the rats was isolated and fixed with 10% formalin. After 7 days, decalcification and dehydration treatment were carried out. Two months after the end of the experiment, the left femur of each group of the three rats was selected for pathological section observation. Each rat was selected from five locations near the left femur for photograph analysis. The H and E staining of bone tissue slices showed that in the model group, the trabecular bone was thinned, broken, scattered, and atrophied, whereas the trabecular bone of rats in normal group was regularly arranged and distributed densely and uniformly. This suggested that the microstructural changes of trabecular bone in OP rats caused by RA were related to bone quality. Compared with the model group, the trabecular bone in each medication-administered group was compact, smooth in surface, and less interrupted. This showed that TFHT could effectively prevent the changes of bone microstructure and the reduction of bone mass caused by RA and improve the bone strength.


  Conclusion Top


To sum up, TFHT can greatly improve the general condition of OP in rats induced by RA. It can effectively prevent bone loss, inhibit bone resorption, promote bone formation, improve the microstructure, prevent the damage to gonads and compensatory hyperplasia of the spleen in rats caused by RA, and improve the negative balance of bone metabolism. Therefore, TFHT was found effective in treating OP in rats induced by RA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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