Metabolic bone disease is related to patients with chronic liver disease,represented as osteoporosis, osteopenia, or osteomalacia, which increase bone pain and fracture risk (Collier 2007; Rouillard and Lane 2001). The pathogenesis of hepatic osteodystrophy (HOD) is complexed and likely is multifactorial, after its formation, HOD is hard to treat (Liu et al. 2018; Moschen et al. 2005; van der Merwe et al. 2003). To allow an early diagnosis and find the appropriate treatment plan, it is important to understand the mechanisms causing HOD. Therefore, our aim consisted in building a rat model mimicking the human situation and easily transferable to other mouse strains for looking into specific proteins or genes.
In 1948, scholars suggested that TAA has hepatotoxicity, can lead to liver fibrosis, and even produce liver tumors in a certain drug concentration and within a certain time, at the same time, the researchers did not find that TAA had an effect on other organs, and suggested that the extent to which certain organs were affected may not be discovered, and further research is needed(Fitzhugh and Nelson 1948). Then, TAA has been shown to cause cholangiocarcinoma(Gupta 1955). Some scholars have also found that TAA not only damages the liver of animals, but also affects the kidneys, brain, spleen, and bone of animals(Al-Bader et al. 2000; Kleinfeld 1957; Lassila and Virtanen 1984b; Saran et al. 2004). When the liver of animal model was treated by TAA, the enzyme metabolism of the liver, protein, adipose, amino acid and the messenger RNA were changed to varying degrees compared with the normal group (Dweep et al. 2017; Fontana et al. 1996; Nozu et al. 1992; Okuyama et al. 2005; Waters et al. 2005). However, most scholars are concerned about the role of TAA in the liver and its mechanism. Few scholars are concerned about the bone damage caused by TAA.
Our data underline that rats with severe liver diseases resulting in osteopenia, especially weight-bearing bones. In this study, the increased concentration of AST and ALT represented severe liver injury, in addition, the increased concentration of UA, UREA, CREA, and TBA represented severe kidney injury, which correlates with other studies (Al-Hashem et al. 2019; Jeong et al. 2015; Waters et al. 2005). We also found the decreased concentration of TP and increased concentration of ALP, which indicated liver disease(Li et al. 2017). The levels of rat serum ALT, AST, and ALP exposed to 200 mg/Kg TAA and 400 mg/Kg TAA group were significantly increased, which indicated that the liver cell membrane was impaired and the release of them into blood was increased after TAA administration. Our data showed serum calcium concentration of 200 mg/Kg TAA and 400 mg/Kg TAA group was decrease compared with control group, the reason of decreased serum calcium is associated with the progression of cell injury since alterations in cell signaling play a determinant role in the toxicological processes (Diez-Fernandez et al. 1996). Serum phosphorus is primarily in the form of inorganic phosphate, which is maintained within the physiological range by regulation of bone formation, dietary absorption, and renal excretion, as well as equilibration with intracellular stores, long-standing phosphorus deficiency will increase risk of osteomalacia (Takeda et al. 2012), then, the concentration of serum phosphorus in our data was decrease compared TAA-treated group with control group, which is as expected in other literature. LDH is an insensitive index of all types of hepatic necrosis except hypoxic (Burke 1978), the level of LDH was not increase compared TAA-treated group with the control group in our studies. In our rats model, TAA-treated animals developed severe liver fibrosis as demonstrated by Masson staining which correlates with other studies (Okuyama et al. 2005; Tuñón et al. 2009), In the Masson-stained picture of the liver, the fibrosis of liver was indicated the pathological sections stained with blue dye, the more blue staining, the more severe the liver fibrosis. In conclusion, we successfully established a rat model of liver injury caused by TAA, so we examined the changes in the bones of the rats by µCT, we obtained 3D reconstruction data of bone area that we interested. Firstly, we can see the change of weight level in three group, a decrease dramatically of weight in 200 mg/Kg TAA and 400 mg/Kg TAA group was observed, which indicated TAA-treated rats was affected by intraperitoneal injection of TAA, Neal have reported that thiono-sulfur compounds exhibit toxic properties in mammals, these effects include bone marrow depression, liver damage (Neal and Halpert 1982). Pauli Virtance considered an increase in osteoclastic resorption in the alveolar bone around the occlusal stressed tooth simultaneously with a horizontal bone loss (Virtanen and Lassila 1986). Secondly, µCT analysis of the bone revealed that the bone structure significantly changed in TAA-treated rats, in the cortical bone, BMD were not affected, strong decreases in BV, TV, BS, and TS were observed, at the same time, most significant changes were observed in the trabecular bone, where reduced BV and TV came along with decreased BS and TS. Furthermore, a decrease in the diameter of femur was observed on the Fig. 5, especially 400 mg/Kg TAA group compared with control group. In addition, a decrease in the cortical bone thickness was observed on the Fig. 7 when 400 mg/Kg TAA group compared with control group. In the last, force analysis of femur represented gradually down trend form normal group to TAA-treated group, TAA administration is leading to bone fragility and increase risk of fracture. From µCT data, we can see that the main part of skeletal injury caused by TAA is in cortical bone, but the effect of TAA on trabecular bone is not so obvious, the administration time of TAA may too short because of only had five intraperitoneal injections. Therefore, our results showed TAA administration have effect on bone metabolism of SD rats, it may relate to liver injury, but no literature suggests that acute liver injury can quickly affect bone metabolism. In this rat model, significant changes in bone metabolism were observed only 20 days, it can quickly build a rat model to represent bone disease for studying mechanism of liver damage. The liver is a multifunctional organ that occupies a key position in the modulation of protein, lipid, and carbohydrate metabolism, and it plays a significant role in mineral metabolism and growth (Nussler et al. 2014), In the literature, there has been a convergence of evidence suggesting bone loss is the primordial bone disorder when found patients was in the early stage of hepatic disease (Guanabens et al. 2013), Therefore, after we speculate that TAA is injected intraperitoneally, TAA or TAA complex will directly damage the bones of rats through the rats' own metabolism. Osteoblasts have affected on osteoclast formation, differentiation, or apoptosis through several pathways, such as OPG/RANKL/RANK, RANKL/LGR4/RANK, Ephrin2/ephB4, and Fas/FasL pathways (Chen et al. 2018), when patients with chronic liver disease were observed, the literature demonstrate that it is associated with alterations in OPG/RANKL pathway, which could modulate bone loss (Moschen et al. 2005; Orsini et al. 2013). In addition, in rats with TAA-induced or carbon tetrachloride-induced cirrhosis, there were reduction in bone volume and were histologically similar to osteoporosis in humans, Atsushi Nakano suggested chronic parenchymal liver injury itself causes osteoporosis due to a combination of low bone formation rates and high resorption rates, the principal pathogenesis of HOD seems to be intestinal Ca malabsorption due to lower serum albumin and villous atrophy, serum levels of vitamin D metabolites have little influence on the pathogenesis of HOD (Nakano et al. 1996), however, as healthy postmenopausal women, vitamin D supplementation resulted in a small but significant improvement in hip bone density, did not significantly reduce hip fracture, while increased the risk of kidney stones (Jackson et al. 2006). Although vitamin D has a critical effect on liver injury and it is related to 25-hydroxyvitamin D that is the major circulating metabolite of vitamin D, the association in liver injury of TAA-treated and bone disease suggest that further research is necessary to clarify potential links. Osteoporosis was associated with primary biliary cirrhosis (PBC) and was a risk factor for vertebral fracture (Guanabens et al. 2010), further studies that the links of TAA, PBC, and vertebral fracture will be necessary to establish their role in the pathogenesis of liver injury. Then again, some studies suggested vitamin K2, Zinc and selenoprotein P have important effect on bone metabolism (Lwamoto et al. 2011; Pietschmann et al. 2014; Sun et al. 2011), insulin-like growth factor-1 (IGF-1) is a main determinant of low cortical but not trabecular bone mass (Liu et al. 2018). We guess IGF-1 associated with metabolic bone disease.