The damaging action of chronic smoke exposure on the skeleton and its possible mechanisms have been extensively studied by us and reported in details [24
]. We demonstrated that the BMD and BMC of the femur neck and lumbar vertebrae were lower in 4-month smoke exposed rats than in controls. Moreover, lower serum osteocalcin concentration and activity of b-ALP and greater TRACP 5b level were noted in the passive smoking group, consistent with the results of previous report [21
]. These results indicated that smoke exposure suppressed bone formation and increased bone resorption. In addition, significant lower trabecular bone volume, trabecular thickness, trabecular number, cortical thickness, bone formation rate and osteoblast surface per bone surface and significant greater trabecular separation and osteoclast surface per bone surface were found in the passive smoking groups. Taking into account the observations made in the present study and the previous reports [24
] the damaging action of smoke exposure on the skeleton might result from its direct action. This mechanism involved stimulation of bone resorption and inhibition of its formation through a direct influence on both differentiation and activity of osteoblastic and osteoclastic bone cells.
Chinese herbal medicine has been widely used for thousands of years in the treatment of fracture and joint diseases. EPF was found to have a preventive effect on osteoporosis by ovariectomy rat models [16
] and exert a beneficial effect on preventing bone loss for postmenopausal women [33
]. Icariin is a major constituent of flavonoids isolated from the herb Epimedium
. The previous studies reported that icariin can stimulate proliferation and differentiation of human osteoblasts by increasing production of BMP-2, BMP-4, NO synthesis, promoting the ALP activity and type I collagen expression, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions [17
]. Meanwhile, icariin also was found to can inhibit osteoclast formation by RANKL and macrophage-colony stimulating factor, and inhibit osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis[37
However, the beneficial influence of EPF on the skeleton at the exposure to cigarette smoke has not been shown until now. The enhanced serum osteocalcin concentration and b-ALP activity due to EPF indicated an enhanced osteoblastic activity in the whole skeleton. Our findings about TRACP 5b, would indicate that EPF can prevent the smoke-induced increased osteoclast activities. The pattern of change in bone turnover was consistent with the findings from previously published study on EPF in an ovariectomized rat model [39
]. Icariin, one of the major flavonoids in EPF (77%), has been reported to stimulate proliferation of rat bone marrow stromal cells and increase the number of colony unit forming-fibroblasts staining positive for alkaline phosphatase in a dose-dependent manner, whereas the alkaline phosphatase activity, OC secretion, and calcium deposition level of rat bone marrow stromal cells were also increased by icariin in a dose-dependent manner, suggesting a potential anabolic effect of icariin on bone [40
]. Furthermore, Genistein and Daidzein, the other two flavonoids within EPFs (23%), have also been reported to have a stimulatory effect on protein synthesis and on alkaline phosphatase release by various types of osteoblast cells in vitro [41
]. Thus, the beneficial effect of EPF consisted in the stimulation of osteoblast activity as well as the reduction of smoke-induced bone resorption.
Kim BS et al. [44
] showed that nicotine suppressed osteoblast proliferation and inhibited the expression of some key osteogenic (TGF-β1, BMP-2) and angiogenic mediators (PDGF-AA, VEGF) in the in vitro experimental model. In addition, Ma
L et al. [45
] reported that Calcium accumulation, ALP activity, and mRNA levels of ALP, bone sialoprotein (BSP), collagen type I α 1 (Col1αI), and runt-related transcription factor 2 (Runx2) were significantly decreased by treatment with nicotine, while osteocalcin transcripts decreased by treatment with nicotine. Thus, nicotine may interact with EPF since nicotine also regulates proliferation and differentiation of human osteoblasts and modulates bone metabolism-associated gene expression in osteoblast cells [44
In this study, the 8-month rats were in the final phase of bone mass formation (bone mass consolidation) and its metabolic processes in the bone slow down. The cellular mechanism behind the anabolic effect of EPF has not been established clearly yet. EPF-treatment may induce a positive bone balance in the remodeling cycle, but this has not been shown directly by histomorphometry. Alternatively, part of the bone anabolic effect of icariin or other EPF components may result from the induction of bone formation by modelling. Such an effect has been demonstrated already to be part of the bone anabolic action of parathyroid hormone by Lindsay and Dempster in iliac crest biopsies [47
], and the same may be true for icariin. Our study showed that EPF can succeed to bring the histomorphometric parameters and biomechanical properties back to control levels and prevent from all of the adverse effects of smoke exposure on bone. This study also showed that EPF can exhibit a beneficial effect on preventing bone loss in passive smoking rats, which was shown by maintained BMD and BMC at the femur neck and lumbar vertebrae in the EPF treatment group compared with significantly decreased BMD and BMC in the passive smoking group. In addition, it is important to note that the higher mean values of the densitometric parameters were found in the EPF group receiving passive smoking than in the passive smoking group or the control group. The alternative explanations for the increase in BMD may include induction of periosteal and endocortical bone modeling (increased cortical thickness), induction of trabecular micro modeling and prevention of marrow cavity expansion, etc. These results may suggest that EPF via its positive effect on bone might also somewhat induce bone modeling.
The influence of EPF on the bone mineral status and bone turnover during the exposure to cigarette smoke somewhat differed depending on EPF dose. Additional daily consumption of 75
mg EPF/kg body wt was noted to be sufficient in this regard; however, the daily dose of 150 or 300
mg EPF/kg body wt may offer stronger protection. The osteoblast surface and many static parameters of bone geometry indicated that the bone formation was dose dependent. The results suggested that there was a bone formation inducing component in EPF which induced a bone anabolic response independent of its effect on bone resorption. As mentioned above this could be in the form of induction of bone modeling, which was independent of bone resorption.
The effects of EPF on bone have been reported [16
]. Thus, this study has not a series of rats treated only with EPF (without smoking). Considering how this medication could be used in the future, it would be necessary to include an additional control group that is kept under normal environment without smoke but receives EPF. That is exactly what we are planning to do in the near future.
Though the present study demonstrate that EPF has a protective effect on the skeleton in smoke-exposed rats, it is not clear whether EPF has a protective effect on the cardio-respiratory effects of smoking and the increased risk of cancer up to now. Even if the smoker can inhibit their bone loss by taking this medicine, smoking also is not ok. According the 2004 Surgeon's General Report, every smoker lives on average 13–14
years less than non-smokers. Smoking also causes cancer, damages organs, and weakens the immune system. In addition, smoking can also harm those nearby, in terms of second-hand smoke. Therefore, it would still be recommended to quit smoking or never initiate it in the first place.