Possible therapeutic effects of berberine on bone damage in high-fat diet-induced obese rats

Background Obesity has a negative impact on bone health; thus far, no specific pharmacotherapy has been approved. Berberine has shown improvements in osteoporosis in some animal models. However, it remains unknown whether berberine ameliorates obesity-induced bone damage. This study aims to evaluate the effect of berberine on bone damage in high-fat diet (HFD)-induced obese rats. Methods Male Sprague-Dawley rats were randomly assigned to a normal control diet (ND) group or HFD group. After the HFD induced obesity, the models were successfully established. Then, these rats were randomly divided into the HFD + berberine (HB) group or HFD group and were intraperitoneally administered berberine or an equivalent volume of DMSO, respectively, once a day for another 10 weeks. Micro-CT and three-point bending tests were conducted to evaluate bone microstructure and biomechanics. Serum was collected for the detection of P1NP, CTX-1, calcium (Ca) and phosphorus (P), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). HE staining was performed to observe the number of trabecular and bone marrow adipocytes. Results HFD-induced obese rats displayed decreased biomechanical properties, such as maximum load, maximum fracture load, ultimate tensile strength, stiffness, and energy absorption, compared with the ND rats (p<0.05). However, berberine attenuated bone damage, including maximum load, maximum fracture load, and stiffness, in the HB group compared with the HFD group (p<0.05). Trabecular bone markers were decreased in the HFD group compared with the ND group (p<0.05). All parameters were improved, as shown by micro-CT and HE staining, in the HB group compared to the HFD group. Cortical bone markers were not significantly different among all groups. Moreover, CTX-1, TNF-α, IL-1β and the number of adipocytes in bone marrow were significantly increased in HFD-induced obese rats. After treatment with berberine,

Technology Co. (Beijing, China). All experimental rats were housed in sterilized cages under conditions of 12 h light/dark cycles, 50% humidity, and controlled temperature (22-24°C) and given free access to food and water. After acclimatization for 1 week, rats were randomly assigned to one of two diets: normal control diet (ND group, n=8) (10% kcal fat) or high-fat diet (HFD group, n=30) (60% kcal fat, Beijing Keao Xieli Feed Co. Ltd., Beijing, China). Body weight was tested weekly. After 12 weeks, HFD-induced obese models were successfully established, when the body weight and fat percentage of rats in the HFD group were significantly higher than those in the ND group [15]. Then, these rats were randomly divided into the HB group (HF diet + berberine (Sigma-Aldrich, St. Louis, MO, USA), n=8) or the HFD group (HF diet, n=8) and were intraperitoneally administered berberine at Statistical analysis Data were analysed by SPSS 22.0 software and are expressed as the mean ± standard deviation (SD).
Means were compared using unpaired Student's t-tests for comparisons between two groups and oneway ANOVA for comparisons among multiple groups. A two-tailed P value <0.05 was considered significant.

Rat model of HFD-induced obesity
The obese rat model was successfully established when the body weight of rats in the HFD group increased more than 20% compared with that of rats in the ND group. In our study, a HFD led to a higher body weight in rats at the end of the experiment than a ND (Fig. 1b). Body weight in the HFD group increased 26.9% compared with that in the ND group at the end of 12 weeks (Fig. 1c). The total fat percent in the HFD group increased significantly compared with that in the ND group (Fig. 1d).
There was no difference in total muscle between the HFD group and the ND group (Fig. 1e).
The effect of berberine (BBR) on P, Ca, bone formation markers, bone resorption markers, and inflammatory factors IL-1β and TNF-α in rats There was no difference in serum P or Ca between the ND group and HFD group before treatment with BBR (12 weeks). There was also no difference in serum P or Ca among the three groups after treatment with BBR (22 weeks) ( Fig. 2a-d).
In contrast, the serum levels of the bone formation marker P1NP were lower in the HFD group than in the ND group, although the difference was not significant (p = 0.7809 (12 weeks), p = 0.2637 (22 weeks)), and were significantly higher in the HB group than in the HFD group (p = 0.0101) (Fig. 2e-f).
In contrast, the serum levels of CTX-1, a marker of bone resorption, were elevated in the HFD group compared with the ND group (p = 0.1172 (12 weeks), p = 0.014 (22 weeks)) and reduced in the HB group compared with the HFD group (p = 0.0784) (Fig. 2g-h).
The serum levels of IL-1β and TNF-α were higher in the HFD group than in the ND group (p = 0.0286, p = 0.0387 at 12 weeks; p = 0.0026, p = 0.0014 at 22 weeks) and significantly lower in the HB group than in the HFD group (p = 0.0002, p = 0.0139).
BBR improved bone biomechanical properties after bone damage in HFD-induced obese rats The results of the three-point bending test in the femur samples showed that the biomechanical properties, such as maximum load (p = 0.0003), maximum fracture load (p = 0.0024), stiffness (p = 0.0028), energy absorption (p = 0.0029), and ultimate tensile strength (p = 0.003), were significantly decreased in the HFD-induced obese group compared with the ND group. The maximum load (p = 0.0062), maximum fracture load (p = 0.0226) and stiffness (p = 0.0171) were significantly enhanced in the HB group compared with the HFD group, but ultimate tensile strength, energy absorption, and elastic modulus showed no significant differences between the HB group and HFD group. (Fig. 3a-f).

The effect of BBR on the trabecular number and the number of adipocytes
The results of HE staining in the left tibia samples showed that the trabecular number was decreased in the HFD-induced obese rats compared with the ND rats (p < 0.01), while those in the HB group had an increased trabecular number compared with those in the HFD group (p = 0.0546) ( Fig. 5a-d). We also found that the number of adipocytes in the HFD group was increased compared with that in the ND group (p < 0.01), while it was significantly decreased in the HB group compared with the HFDinduced obese group (p < 0.01) (Fig. 5a-c and e).

Discussion
In this study, we investigated bone health in HFD-induced obese male rats and the effect of berberine on these rats. In accordance with recent studies, our results show that obesity significantly reduces bone microstructure and bone strength, and the major finding of this study was that berberine can alleviate this damage to some extent.
Obesity is a global health issue associated with significant morbidity and mortality, which independently increases the risk for chronic diseases such as type 2 diabetes mellitus (T2DM), cardiovascular diseases, hypertension, stroke, and some types of cancer [16]. In addition, its negative impact on bone damage has been recently reported. Fewer studies on osteoporosis have been conducted in males than in females. Recent studies indicated that visceral adipose tissue was also a risk factor for osteoporosis in men [4,17] and that obese men were more likely to have vertebral fracture than men of normal weight [5]. Another study indicated that obesity induced by a HFD aggravated bone loss in the cancellous bone compartment, with a greater loss in males than females [18]. Our current study also focused on male rats, and bone damage was observed in HFD-induced obese male rats. Trabecular bone loss and bone strength declined. Tb. vBMD, Tb. BV/TV, and Tb. N were decreased, and Tb. Th, Tb. Sp and SMI were increased in the HFD-induced obese group compared with the ND group. The maximum load, maximum fracture load, stiffness, energy absorption, and ultimate tensile strength were significantly decreased in the HFD-induced obese group compared with the ND group. However, cortical bone parameters remained unchanged in our study. Consistent with Cao et al [19], obesity induced by a high-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia. The finding that cancellous rather than cortical bone is affected in HFD-induced obesity is not surprising. In general, cancellous bone is more responsive than cortical bone to diet or drug treatments, physiological status, or ageing because cancellous bone undergoes more active remodelling than cortical bone due to its larger surface-tovolume ratio [20]. In our study, bone strength decline in HFD-induced obesity was attenuated significantly by treatment with berberine, as shown by the three-point bending test. Yogesh, H. S. indicated that berberine chloride exerted protective effects on bone in an osteoporotic rat model induced by ovariectomy [10], and Xu et al demonstrated that berberine significantly increased femur load and stiffness in glucocorticoid-treated animals [11], which were consistent with our study. Xie et al showed that berberine increased femoral BMD, BV/TV, Tb. N, and Tb. Th as well as reduced Tb. Sp and SMI in rats with diabetic osteoporosis induced by streptozotocin and a high-fat diet [13].
Trabecular bone loss was also attenuated in our study, but the difference was not significant, as shown by micro CT and HE staining.
The results from the present study demonstrated increased inflammatory cytokine levels and bone marrow adiposity in HFD-induced obese rats, which might not be good for skeletal strength or for optimal function of the bone remodelling unit [21]. Osteoblasts and adipocytes are derived from a common multipotential mesenchymal stem cell [22]. Marrow stromal cells isolated from postmenopausal osteoporotic patients express more adipocytic differentiation markers than those with normal bone mass [23] and are more likely to enter an adipocyte differentiation programme than an osteoblast one [24]. Peroxisome proliferator-activated receptor gamma 2 (PPARg2), an essential factor in adipocyte differentiation, can inhibit osteoblast differentiation [25]. Fat in bone marrow might not only suppress osteoblastogenesis but also promote bone resorption because marrow adipocytes, much like fat cells elsewhere, secrete inflammatory cytokines [26]. Obesity is associated with a chronic inflammatory response, abnormal cytokine production, and activation of inflammatory signalling pathways, which are involved in and responsible for the development of obesity-related diseases [27]. Proinflammatory cytokines, including TNF-α, IL-1, and IL-6, are key mediators in the process of osteoclast differentiation and bone resorption. These proinflammatory cytokines are capable of stimulating osteoclast activity through the regulation of the RANKL/RANK/OPG pathway [28,29]. Normal bone development can occur in mice lacking IL-1 and TNF genes [30]. Interestingly, overexpression of soluble TNF-α decoy receptor [31] or ovariectomy did not cause bone loss.
However, blocking the action of IL-1 with an IL-1 receptor antagonist or the signalling of TNF-α with a TNF-binding protein decreased osteoclast formation and bone resorption in ovariectomized mice [32].
Furthermore, superoxide-driven oxidative stress, a common factor in obese adipose tissue, has been shown to stimulate osteoclastogenesis, bone matrix degradation, and bone resorption [33]. In our study, the circulating bone resorption marker CTX was significantly increased in obese animals compared with the ND animals, which confirmed that bone resorption was increased. After treatment with berberine, TNF-α and IL-1β levels and the number of adipocytes in bone marrow were significantly decreased in the HB group compared with the HFD-induced obese group. Berberine significantly increased the circulating bone formation marker P1NP. There was no significant difference in the bone resorption marker CTX. Furthermore, the number of rats in the experiment was limited, so this difference may become statistically significant if the sample size is increased. These results indicate that the inhibitory effects of berberine on inflammation and bone marrow adipocytes could promote osteoblastogenesis and inhibit bone resorption, which could be one mechanism by which berberine prevents obesity-induced osteoporosis.
There were some limitations in this study. One limitation was that the present study was conducted in a rodent model and not in humans. Another limitation was that the mechanism by which berberine alleviates bone damage was not investigated at the molecular and genetic levels. In the next step, we will conduct in vivo experiments to further elucidate the protective mechanism of berberine in alleviating bone damage induced by obesity at the molecular and genetic levels and to provide a theoretical basis for the clinical treatment of bone damage caused by obesity.

Conclusions
In conclusion, the results from the present study suggest that berberine might be a potential therapeutic agent for treating bone damage in HFD-induced obese rats by inhibiting inflammatory factors, reducing bone marrow adiposity and improving bone formation. However, whether the clinical application of berberine could be beneficial in the population needs further confirmation.