Effect and mechanism of Gusong Qianggu Decoction on reducing bone loss in ovariectomized mice by targeting endoplasmic reticulum stress-induced apoptosis of osteocytes
This study aims to investigate the role and mechanism of Gusong Qianggu Decoction(GSQG)in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress(ERS)-induced apoptosis of osteocytes.After the modeling of osteo-porosis in mice with bilateral ovary removal(OVX),60 mice were randomized by the random number method into six groups:sham,model,low-,medium-,and high-dose GSQG(GSQG-L,GSQG-M,and GSQG-H,respectively),and estradiol(E2),with 10 mice in each group.The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the adminis-tration lasted for 3 months.Enzyme-linked immunosorbent assay(ELISA)was employed to determine the serum levels of osteocalcin(OCN),procollagen type Ⅰ N-terminal propeptide(PINP),carboxy-terminal cross-linked telopeptide of type Ⅰ collagen(CTX),and anti-tartarte acid phosphatase 5b(TRAcP-5b).Micro-CT was employed to observe the changes in bone microstructure of the distal femur.Hematoxylin-eosin(HE)staining was employed to observe the morphology of the bone tissue.RT-qPCR was conducted to de-termine the mRNA levels of tibial stem osteogenesis-associated genes[type Ⅰ collagen(Col-Ⅰ),alkaline phosphatase(ALP),Runt-related transcription factor-2(Runx2),bone sialoprotein(BSP),and OCN]and bone-breaking related genes[tartrate-resistant acid phosphatase(TRAP),nuclear factor-activated T cell 1(NFATc1),and cathepsin K(CATK)].TUNEL staining and immunohisto-chemistry were employed to detect the apoptosis of osteoblasts.Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78(Grp78),protein kinase RNA-like endoplasmic reticulum kinase(PERK),phosphorylated PERK(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2α),phosphorylated eIF2α(p-eIF2α),inositol-requiring en-zyme 1 alpha(IRE1α),phosphorylated IRE1α(p-IRE1α),and activating transcription factor 6(ATF6)in the proximal tibial bone tissue.The results showed that GSQG significantly recovered the levels of OCN,PINP,TRAcP-5b,and CTX in the serum of ovariec-tomized mice,and Micro-CT showed that GSQG improved the bone microstructure of distal femur in a dose-dependent manner.Com-pared with the model group,GSQG widened and increased the bone trabeculae,restored the reticular structure with neat arrangement and enlarged interstitial gaps,and reduced the number of TUNEL-positive cells(P<0.05,P<0.01).Furthermore,GSQG down-regu-lated the expression levels of cysteine aspartate protease-3(caspase-3)and factor Bcl-2-associated X protein(Bax)(P<0.05,P<0.01)and up-regulated the expression level of Bcl-2(P<0.05,P<0.01).The GSQG groups showed up-regulated mRNA levels of Col-Ⅰ,ALP,Runx2,BSP,and OCN(P<0.01)and down-regulated mRNA levels of TRAP,NFATc1,and CATK(P<0.05,P<0.01).In addition,GSQG,especially GSQG-H,down-regulated the protein levels of Grp78,p-PERK,p-eIF2,p-IRE1α,and ATF6(P<0.05,P<0.01).In conclusion,GSQG can inhibit the apoptosis of osteocytes by inhibiting the Grp78/PERK/eIF2α/IRE1α/ATF6 signaling pathway in the proximal tibia tissue,thus reducing bone loss in ovariectomized mice.
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