Therapeutic Effect of Guizhi Shaoyao Zhimutang on Osteoporosis in Rats Based on RANKL/OPG / 中国实验方剂学杂志

ABSTRACT

Objective:To investigate the therapeutic effect of Guizhi Shaoyao Zhimutang （GSZT） on the osteoporosis （OP） in SD female rats induced by ovariectomy （OVX） combined with glucocorticoid injection and its related mechanisms. Method:The rats were divided into a sham operation group and an experimental group for OVX. One week later， the experimental rats were divided into an OP model group， a calciferol （positive control， 0.1 mg·kg^-1） group， and low（0.8 g·kg^-1）， medium（1.6 g·kg^-1）， and high-dose （3.2 g·kg^-1） GSZT groups. Except for those in the sham operation group， the rats received an intramuscular injection of dexamethasone （1 mg·kg^-1） twice per week for six weeks. After the rats were treated correspondingly for eight weeks， the rats were sacrificed for thighbone sample collection. The computer microtomography （Micro CT） was used to analyze the parameters of bone mineral density （BMD）， bone volume （BV）， tissue volume （TV）， structure model index （SMI）， connectivity density （Conn.D）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， trabecular pattern factor （Tb.Pf）， and trabecular separation （Tb.Sp）. In addition， hematoxylin-eosin （HE） and Masson staining was used for the histopathological examination of the thighbone. Tartrate-resistant acid phosphatase （TRAP） staining was used to analyze osteoclasts in bone tissues. The enzyme-linked immunosorbent assay （ELISA） was used to determine matrix metalloproteinase-9 （MMP-9）， cathepsin K （CTSK）， and TRAP5b in bone tissues， and Western blot was carried out to determine receptor activator of NF-kappaB ligand （RANKL）， osteoprotegerin （OPG）， alkaline phosphatase （ALP） in bone tissues. Result:Compared with normal group， the number of bone trabeculae in cancellous part of OP model rats decreased significantly， the spacing widened， the thickness became thinner， and the continuity was poor. Compared with model group， GZST medium and high-dose groups could improve the changes of femoral bone trabecular cancellous bone. Masson staining showed that compared with the normal group， the growth plate thickness of the distal femur epiphysis was thinner and the area of new bone was smaller in the OP model group， while the reduction of new bone was inhibited in the high dose group of calcitol and GZST. TRAP staining results showed that the number of osteoclasts in OP group was significantly increased compared with the normal group， and the number of osteoclasts in medium-high dose GZST group was reduced. Micro CT results showed that compared with normal group， BMD， BV， Tb.N， Tb.Th and Conn.D of OP rats were decreased， SMI and Tb.Sp were significantly increased（<italic>P</italic><0.01）. Compared with model group， medium and high dose of GZST could improve the degree of osteoporosis in OP rats， BV， Tb.N were significantly increased， SMI， Tb.Sp were significantly decreased （<italic>P</italic><0.01）. ELISA results revealed that compared with the sham operation group， the OP model group showed decreased ALP content （<italic>P</italic><0.01） and increased MMP-9， TRAP5b， and CTSK （<italic>P</italic><0.01）. Compared with the OP model group， the high-dose GSZT group showed increased ALP content （<italic>P</italic><0.01） and decreased MMP-9， TRAP5b， and CTSK （<italic>P</italic><0.01）. Western blot results demonstrated that compared with the OP model group， the calciferol group and the high-dose GSZT group showed inhibited expression of the RANKL protein （<italic>P</italic><0.05）， and increased OPG expression （<italic>P</italic><0.05）. Conclusion:The findings suggest that GZST has a potential therapeutic effect on OP caused by OVX combined with glucocorticoids in rats， and the underlying mechanism is associated with the regulation of RANKL/OPG in bone tissues. This study is expected to lay a preliminary foundation for its clinical application and modern preparation development.