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Journal of Chinese Integrative Medicine ›› 2010, Vol. 8 ›› Issue (2): 173-180.doi: 10.3736/jcim20100213

• Original Experimental Research • Previous Articles     Next Articles

Effects of Qishe Pill on vertebral hyperostosis induced by upright posture in rats

Qin Bian , Qian-qian Liang, Wei HOU, Yong-jian Zhao, Sheng Lu, Yong-jun Wang,Qi Shi   

  1. Institute of Spine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Accepted:2010-01-07 Online:2010-02-20 Published:2010-02-15
  • Contact: Yong-jun Wang E-mail:yjwang88@hotmail.com

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Objective

To observe the effects of Qishe Pill, a compound traditional Chinese herbal medicine, on lumbar vertebral bone formation induced by long-time upright posture in rats and to investigate the potential mechanism.
Methods

Thirty SD rats were randomly divided into normal control group, untreated group and Qishe Pill group. The rats in normal control group received no treatment and were raised in normal cages. The rats in untreated group and Qishe Pill group were cut off forelimbs by operation so as to be forced to adopt an upright posture for 8 months to induce hyperostosis. Rats in the Qishe Pill group were intragastrically administered with Qishe Pill at a dose of 5 g/(kg · d) for 1 month. All rats were sacrificed at the 9th month after surgery and all lumbar vertebrae were harvested for detection. Safranin O/fast green staining and picrosirius red staining were used to observe pathological changes. Expressions of type Ⅰ collagen (ColⅠ), type Ⅹ collagen (ColⅩ), vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) in the 5th lumbar vertebra (L5) were detected by immunohistochemical method. Expressions of type Ⅰ collagen α2 (Col1α2), type Ⅹ collagen α1 (Col10α1), TGF-β1, and VEGF and runt-related transcription factor 2 (Runx2) mRNAs in L1-L3 were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction.
Results

Safranin O/fast green staining showed that in the untreated group, non-matrix components increased at marginal lumbar vertebra and intervertebral disc junction, and hyperostosis appeared. However, no obvious change was observed in the normal control group. Non-matrix components decreased at the same location in Qishe Pill group as compared with the untreated group. Picrosirius red staining showed compact collagens at marginal lumbar vertebra and intervertebral disc junction in the normal control group, however, ColⅠ and ColⅩ increased at the same location in the untreated group. In the Qishe Pill group, it showed more compact collagens, especially ColⅠ. Compared with normal control group, expressions of ColⅩ, VEGF and TGF-β1 were increased at marginal lumbar vertebra and intervertebral disc junction in the untreated group. ColⅩ and VEGF expressions decreased in the Qishe Pill group as compared with the untreated group. Col10α1 and Runx2 mRNA expressions were down-regulated by Qishe Pill (P<0.01).
Conclusion

Qishe Pill may delay hyperostosis at marginal lumbar vertebra and intervertebral disc junction, which may be related to reducing type Ⅹ collagen and Runx2 expressions.

Key words: Qishe Pill, Hyperostosis, Fibrillar collagens, Vascular endothelial growth factor, Transforming growth factorPI, Runt-related transcription factor 2, Rats

Table 1

Sequences, accession numbers and product lengths of rats’ β-actin, Col1α2, Col10α1, VEGF, TGF-β1 and Runx2 primers"

Gene Forward primer 5′-3′ Reverse primer 5′-3′ Accession number Product length (bp)
β-actin GGAGATTACTGCCCTGGCTCCTA GACTCATCGTACTCCTGCTTGCTG NM_031144 150
Col1α2 TCCTGGCAATCGTGGTTCAA ACCAGCTGGGCCAACATTTC NM_053356 133
Col10α1 TTCACAAAGAGCGACAGAGA TCAAATGGGATGGAGCA NM_053819 143
VEGF TGGACCCTGGCTTTACTGCTG GGCAATAGCTGCGCTGGTAGA NM_031836 127
TGF-β1 TGCGCCTGCAGAGATTCAAG AGGTAACGCCAGGAATTGTTGCTA NM_021578 82
Runx2 CCATAACGGTCTTCACAAATCCT TCTGTCTGTGCCTTCTTGGTTC XM_001066762 99

Table 2

Reactive system for real time fluorescent quantitation"

Content Volume （μL）
Master mix 10
RNase-free water 7
Forward primer 1
Reverse primer 1
cDNA 1
Total volume 20

Figure 1

Pathologic changes of L5 vertebral edge and disc in coronal plane observed by sirius red staining (Light microscopy, ×200)A: Normal control group; B: Untreated group; C: Qishe Pill group."

Figure 2

Pathologic changes of L5 vertebral edge and disc in coronal plane observed by safranin O/fast green staining (Light microscopy,×200)A: Normal control group; B: Untreated group; C: Qishe Pill group."

Figure 3

Expression of ColⅩ in L5 vertebral edge and disc observed by immunohistochemical staining (Light microscopy, ×200)A: Normal control group; B: Untreated group; C: Qishe Pill group."

Figure 4

Expression of VEGF in L5 vertebral edge and disc observed by immunohistochemical staining (Light microscopy, ×200) A: Normal control group; B: Untreated group; C: Qishe Pill group."

Figure 5

Expression of ColⅠ in L5 vertebral edge and disc observed by immunohistochemical staining (Light microscopy, ×200)A: Normal control group; B: Untreated group; C: Qishe Pill group."

Figure 6

Expression of TGF-β1 in L5 vertebral edge and disc observed by immunohistochemical staining (Light microscopy, ×200)A: Normal control group; B: Untreated group; C: Qishe Pill group."

Table 3

Expressions of Col1α2, Col10α1, VEGF, TGF-β1 and Runx2 mRNAs in conjunctional tissue of vertebral edge and intervertebral discs in rats ($\bar{x}±s$)"

Group n Col1α2 Col10α1 VEGF TGF-β1 Runx2
Normal control 6 10.13±1.08 0.02±0.00 0.67±0.10 0.71±0.10 18.85±3.79
Untreated 6 302.00±50.53** 0.05±0.01** 3.46±0.81** 2.72±0.60** 184.88±23.74**
Qishe Pill 6 292.64±35.62 0.03±0.01△△ 2.47±0.39 2.51±0.14 57.51±1.86△△
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