Tanshinone I Mitigates Steroid-Induced Osteonecrosis of the Femoral Head and Activates the Nrf2 Signaling Pathway in Rats

Steroid-induced osteonecrosis of the femoral head (SIONFH) is a frequent orthopedic disease caused by long-term or high-dose administration of corticosteroids. Tanshinone I (TsI), a flavonoid compound isolated from Salvia miltiorrhiza Bunge, has been reported to inhibit osteoclastic differentiation in vitro. This study aimed to investigate whether TsI can ameliorate SIONFH. Herein, SIONFH was induced by intraperitoneal injection of 20 μg/kg lipopolysaccharide every 24 h for 2 days, followed by an intramuscular injection of 40 mg/kg methylprednisolone every 24 h for 3 days. Four weeks after the final injection of methylprednisolone, the rats were intraperitoneally administrated with low-dose (5 mg/kg) and high-dose (10 mg/kg) TsI once daily for 4 weeks. Results showed that TsI significantly alleviated osteonecrotic lesions of the femoral heads as determined by micro-CT analysis. Furthermore, TsI increased alkaline phosphatase activity and expressions of osteoblastic markers including osteocalcin, type I collagen, osteopontin, and Runt-related transcription factor 2 and decreased tartrate-resistant acid phosphatase activity and expressions of osteoclastic markers including cathepsin K and acid phosphatase 5. TsI also reduced inflammatory response and oxidative stress and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in the femoral heads. Taken together, our findings show that TsI can relieve SIONFH, indicating that it may be a candidate for preventing SIONFH.


Introduction
Osteonecrosis of the femoral head (ONFH), a progressive bone disorder, causes subchondral bone microfractures and significant pain [1]. e age of patients ranged from 20 to 40 years [2]. Approximately 80% of patients receiving surgery during the early stages suffer from femoral heads collapse within 1-3 years [3]. e femoral heads collapse may rapidly progress to hip osteoarthritis that severely impacts hip joint function, ultimately undergoing artificial joint replacement [3]. At present, the long-term therapeutic effect of hip replacement is not usually ideal. erefore, some patients may need to undergo 2-3 replacements, bringing an economic burden to patients and their families. If not treated promptly, patients with ONFH will suffer lifelong disability, which seriously affects the patients' quality of life. ONFH can be categorized as traumatic and nontraumatic. As nontraumatic ONFH, steroid-induced ONFH (SIONFH) was diagnosed in 26.84% of the ONFH patients in China, behind alcohol-induced ONFH [4]. Glucocorticoids are very effective at reducing inflammation, and they are commonly used for the treatment of systemic lupus erythematosus [5], rheumatoid arthritis [6], and multiple sclerosis [7]. Approximately 9%-40% of patients who receive long-term or high-dose (cumulative dose >3 g) glucocorticoid therapy develop ONFH [8,9]. erefore, the strategy to prevent ONFH during glucocorticoid therapy is urgently needed.
Dysregulation of bone homeostasis may be one of the leading causes of SIONFH. Following osteonecrosis, bone resorption mediated by osteoclasts surpasses bone formation by osteoblasts, leading to loss of subchondral trabecular bone. Elevated levels of proinflammatory cytokines have been found in SIONFH patients [10]. It is widely accepted that proinflammatory cytokines interfere with bone homeostasis [11]. In addition, glucocorticoids have a significant influence on oxidative stress [12]. Excessive oxidative stress leads to an imbalance between bone resorption and bone formation, thereby leading to loss of bone mineral density [13]. Multiple drugs exerted therapeutic effects on ONFH through their antioxidative properties [14][15][16], highlighting the significance of oxidative stress in the pathogenesis of ONFH.
Tanshinone I (TsI) is one of the major bioactive flavonoids isolated from Salvia miltiorrhiza Bunge (also known as Danshen in Chinese), which has a tremendous potential for treating cardiovascular diseases [17]. TsI exerts antitumor [18], anti-inflammatory [19], and antioxidative [20] effects on different cell types. Emerging studies have shown that Danshen is a component frequently used in Chinese herbal formulas, including Huo-gu and Fufang Xian Ling Gu Bao, which are effective in preventing SIONFH [21,22]. As one of the most abundant lipophilic compounds in Danshen, TsI has a significant effect on inhibiting osteoclast differentiation [23,24]. As abovementioned, osteoclasts induce bone resorption that plays a pivotal role in the progression of ONFH. erefore, it is worthy of discussion whether TsI has a protective effect against ONFH.
Here, SIONFH was induced in rats by injection of lipopolysaccharide (LPS) and methylprednisolone (MPS), and the rats received TsI treatment for 4 weeks. Subsequently, the effect of TsI on the osteogenesis, osteoclastogenesis, inflammatory response, oxidative stress, and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in the SIONFH rats was analyzed.
is study provides a potential candidate for SIONFH treatment.

Ethics Statement.
Animal experiments were strictly in accordance with the Guide for the Care and Use of Laboratory Animals. e protocol of animal experiments in this study was approved by the Institutional Animal Care and Use Committee of the Heilongjiang University of Chinese Medicine.

Experimental Animals and
Grouping. Male 12-week-old Sprague Dawley (SD) rats were obtained from Beijing HFK Bioscience (China). Rats were maintained at 22 ± 1°C with relative humidity at 45%-55% and a 12/12 h light/dark cycle and allowed free access to food and water. After 7-day feeding adaptation, all the rats were randomized into 4 groups: the control group, the SIONFH group, the SIONFH + low-dose TsI (TsI-L) group, and the SIONFH + high-dose TsI (TsI-H) group.

Induction of SIONFH and Administration of TsI.
Rats in the SIONFH group, the SIONFH + TsI-L group, and the SIONFH + TsI-H group were intraperitoneally injected with lipopolysaccharide (LPS, Sigma-Aldrich, USA) at a concentration of 20 μg/kg body weight on day 0 and day 1 at a time interval of 24 h, followed by intramuscular injection with methylprednisolone (MPS, Pfizer bio, China) at a concentration of 40 mg/kg body weight on day 3, day 4, and day 5 at a time interval of 24 h as previously described [25]. Rats in the control group only received the same volume of normal saline on day 0, day 1, day 3, day 4, and day 5.
TsI (Aladdin, China) was dissolved in DMSO, followed by dilution with normal saline. Rats in the SIONFH + TsI-L group and the SIONFH + TsI-H group were intraperitoneally injected with TsI at a concentration of 5 mg/kg and 10 mg/kg body weight once daily for 4 weeks after the final injection of MPS, respectively. Rats in the control group and the SIONFH group received the same volume of vehicle. After 4 weeks, all the rats were euthanized, and the bilateral femora and serum were harvested for the following experiments.

Micro-CT Scan and Quantitative Analysis.
e femoral heads were subjected to micro-CT scanning using the Quantum GX2 micro-CT (PerkinElmer, USA). Femoral heads on the right side of each group were scanned at 90 kV/ 88 μA of energy with an entire scan length of 5 mm at a 10 μm pixel size. Bone mineral density (BMD), the ratio of bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb. ) were quantified.

Alkaline Phosphatase (ALP) and Tartrate-Resistant Acid Phosphatase (TRAP) Activity Assay.
e activity of ALP in the femoral head and that of TRAP in the serum were measured using the ALP assay kit (Nanjing Jiancheng, China) and the TRAP assay kit (Nanjing Jiancheng, China), respectively, in accordance with their manufacturer's instructions.

Immunohistochemistry (IHC)
Staining. IHC staining was performed using rabbit polyclonal anti-OCN (DF12303) and anti-COL I (AF0134) antibodies (Affinity Biosciences). In brief, the femoral heads were decalcified, embedded with paraffin, and sectioned at a thickness of 5 μm in the coronal plane. e sections were immersed in antigen retrieval solution (1.8% citric acid and 8.2% sodium citrate) for 10 min and then in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. After blocking in goat serum (Solarbio) for 15 min, the sections were incubated overnight at 4°C with anti-OCN or anti-COL I antibody (1 : 50), followed by incubation with horseradish peroxide-conjugated goat anti-rabbit antibody (#31460, ermo Fisher Scientific, 1 : 500) for 60 min at 37°C. e sections were stained with DAB (Solarbio) and counterstained with hematoxylin (Solarbio). e pictures were captured using a microscope (×100) (OLUMPUS, Japan).

Enzyme-Linked Immunosorbent Assay (ELISA).
e concentration of proinflammatory factors including TNF-α, IL-6, and IL-1β in the serum was determined by commercially available ELISA kits (MultiSciences Biotech, China) in accordance with their manufacturer's protocols.

Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-Px), and Chloramphenicol Acetyltransferase (CAT) Activity Assay.
e activity of antioxidant enzymes including GSH-Px, SOD, and CAT in the femoral head was detected using commercial assay kits (Nanjing Jiancheng) following their manufacturer's instructions.

Statistical
Analysis. Data are presented as the mean-± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 8.0.1, GraphPad Software, USA) by the Kruskal-Wallis test (Tb.N) with Dunn's test or one way ANOVA (other data) with Tukey's test. e p values less than 0.05 were taken to be statistically significant.

TsI Facilitates Osteogenesis in the SIONFH Rat Model.
Next, the effect of TsI on osteogenesis in the rat model of SIONFH was explored. e SIONFH group (3.86 ± 1.05 King's unit/g prot) had a low level of ALP activity compared with the control group (11.95 ± 2.53 King's unit/g prot); however, high-dose TsI partly diminished the SIONFHinduced decreases in ALP activity (8.53 ± 1.75 King's unit/g prot) (Figure 2(a)). e expression of osteogenesis makers including OCN, COL I, OPN, and Runx2 was detected by RT-qPCR and western blot analysis. Results exhibited that the mRNA and protein expression levels of OCN, COL I, OPN, and Runx2 were significantly decreased in the SIONFH group (Figures 2(b), 2(c)). High-dose TsI treatments upregulated the mRNA and protein expression of all these osteogenesis makers in the femoral heads of SIONFH rats, while low-dose TsI treatments only upregulated the mRNA expression of Runx2 and the protein expression of OCN, COL I, and OPN (Figures 2(b), 2(c)). Consistently, the increases of OCN and COL I expression by low-dose and high-dose TsI treatments in the femoral heads were also confirmed by IHC staining (Figure 2(d)). ese results indicated that TsI could promote bone formation in the rat model of SIONFH.

TsI Reduces Osteoclastogenesis in the SIONFH Rat Model.
To analyze whether TsI affected osteoclastogenesis in the rat model of SIONFH, the activity of serum TRAP and the expression of CTSK and ACP5 were measured. Elevated activities of serum TRAP were observed in the SIONFH group (the SIONFH group, 47.62 ± 10.30 U/L; the control group, 13.66 ± 3.84 U/L), which could be inhibited by high-dose TsI treatment (24.20 ± 5.42 U/L) (Figure 3(a)). Western blot analysis showed that the expression levels of CTSK and ACP5 in the femoral heads were upregulated after induction of SIONFH, and this action was reversed by high-dose TsI, while low-dose TsI only suppressed the upregulation in ACP5 expressions (Figure 3(b)).
ese results indicated that TsI could inhibit bone resorption in the rat model of SIONFH.
the SIONFH group compared with those of the control group (GSH-Px, 64.82 ± 16.67 U/mg prot; SOD, 28.51 ± 6.24 U/mg prot; CAT, 19.22 ± 4.96 U/mg prot). e decreased activity of those enzymes was partly restored by high-dose TsI treatment (Figures 5(a)-5(c); GSH-Px, 50.24 ± 11.87 U/mg prot; SOD, 19.20 ± 3.99 U/mg prot; CAT, 13.73 ± 3.05 U/mg prot). e proteins involved in the Nrf2 pathway including Nrf2, HO-1, and NQO1 were detected using western blot analysis. e expression of Nrf2, HO-1, and NQO1 was significantly downregulated in the femoral heads after induction of SIONFH, but high-dose TsI treatment reversed the downregulation of expression of these proteins (Figures 5(d)-5(f )). ese results indicated that TsI could reduce oxidative stress and activate the Nrf2 signaling pathway in the rat model of SIONFH.

Discussion
As early as 1932, the detrimental effect of hypercortisolism on bone was identified [26]. With the extensive application of glucocorticoids in clinics, over 50% of patients who receive glucocorticoid therapy develop significant bone loss that results from dysregulation of bone metabolism [27].
bone formation and bone resorption may be one of the most common biological processes in SIONFH. As a traditional herbal medicine in China, Danshen is frequently used to treat some different types of diseases including ONFH. Flavonoids are the major chemical constituents of many traditional herbal medicines, including Scutellaria baicalensis (Huangqin), Sophora flavescens (Kushen), Puerariae Lobatae Radix (Gegen), and Carthamus tinctorius (Hong Hua). Accumulating evidence has demonstrated that lots of flavonoids are effective to treat ONFH, including naringin [28], icariin [29], and luteolin [30]. TsI is one of the main active components in Danshen and its antiosteoclastic effect has been reported in in vitro experiments in earlier studies [23,24]. In the present study, we found that TsI could attenuate SIONFH in a rat model. Moreover, TsI was proved to stimulate osteogenesis and inhibit osteoclastogenesis, indicating its potentials in restoring the balance between bone formation and bone resorption.
Emerging studies have shown that the level of proinflammatory cytokines is elevated in the serum of SIONFH patients [10]. Generally, glucocorticoid treatment is given to suppress inflammation. However, long-term or excess application of glucocorticoids functions as an activator for inflammation. e elevation of proinflammatory cytokines has been found in a mouse model of SIONFH [10,31]. Similar results were found in the rat model of SIONFH in the current study. We also found that TsI exerted an anti-inflammatory effect in the rat model of SIONFH. Bone homeostasis can be critically affected by inflammation. Several earlier studies confirmed that TNF-α, IL-1β, and IL-6 had negative effects on osteoblast differentiation [32][33][34]. TNF-α and IL-1β have also been reported to stimulate osteoclast differentiation [35,36]. erefore, the regulatory role of TsI in bone formation and bone resorption may be ascribed to its anti-inflammatory potential.
Oxidative stress develops when the balance between reactive oxygen species (ROS) generation and ROS scavenging is broken. Antioxidant enzymes are involved in ROS scavenging. It has been reported that oxidative stress is involved in the pathogenesis of multiple bone diseases including ONFH [37][38][39]. ROS has a negative effect on bone homeostasis due to its pro-osteoclastic effects [40]. Oxidative stress causes dysfunction of bone metabolism-related cells including bone mesenchymal stem cells [41], osteoblasts [42], osteoclasts [43], and osteocytes [44]. Glucocorticoids stimulate overproduction of ROS by depletion of antioxidant molecules or inhibition of antioxidant enzymes. Excessive usage of glucocorticoids has been indicated to enhance oxidative stress in an animal model of SIONFH [45]. e function of TsI in inhibiting oxidative stress has been reported in several studies [20]. Consistently, this study observed that TsI exerted its antioxidant potential in SIONFH by increasing the activities of antioxidant enzymes. Considering the important role of oxidative stress in bone homeostasis, the protective effect of TsI against SIONFH may be attributable to its antioxidant potential. In addition, oxidative stress is a pivotal factor in the inflammatory response. Excessive accumulation of ROS aggravates inflammatory response via activation of the nuclear factor-κB (NF-κB) signaling pathway in the development of many diseases [46,47]. erefore, the anti-inflammatory potential of TsI may be associated with its antioxidant potential, which needs to be confirmed in the subsequent works. e transcription factor Nrf2 plays a central role in the maintenance of intracellular redox homeostasis. Stabilized Nrf2 translocates into the nucleus, where it binds to antioxidant-response elements (ARE) in the promoter region of Nrf2 target genes to activate downstream antioxidant enzymes [48]. Accumulating investigations have indicated that the Nrf2 signaling pathway is inhibited in necrotic femoral head tissues and the ONFH model [49]. e present study also confirmed the findings. Earlier findings demonstrated that TsI could activate the Nrf2 signaling [50]. Similarly, we found that TsI activated the Nrf2 signaling in the femoral head of SIONFH models. e increased activities of antioxidant enzymes by TsI may be associated with activation of the Nrf2 signaling. In addition, Nrf2 serves as an enhancer of osteogenesis [51] and a suppressor of osteoclastogenesis [52]. erefore, TsI might promote osteogenesis and restrain osteoclastogenesis through the activation of the Nrf2 signaling pathway in the femoral heads.
It is a limitation that there is no positive control drug in the present study. Although this study has shown the effect of TsI against SIONFH, it is hard to declare to what extent TsI has protective effects. erefore, a comparison of the therapeutic efficacy of TsI with a positive control drug will be needed in our future study.

Conclusion
In summary, our results revealed that TsI could promote osteogenesis and inhibit osteoclastogenesis in the femoral heads of SIONFH rats. Moreover, TsI exerted both antiinflammatory and antioxidant potential in the SIONFH rats. In addition, TsI activated the Nrf2 signaling pathway. ese findings suggest that TsI may be a good candidate for treating SIONFH.
Data Availability e datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.