Protective Role of Panax Notoginseng Saponins in Hydrocortisone-Induced Injury in HBMECs by Promoting APOA1 Expression

To evaluate the protective effect and underlying mechanism of panax notoginseng saponins (PNS) on hydrocortisone (HC)-induced injury in human bone microvascular endothelial cells (HBMECs).HBMECs were isolated from femoral heads resected in total hip arthroplasty and identied by morphology and immunouorescence. HBMECs were injured by hydrocortisone (HC), treated with PNS in different concentrations, and transfected with or siAPOA1 to explore the role of in the process where protected HC-injured HBMECs. The viability and apoptotic rate were determined by CCK-8 and ow cytometry assays, respectively. The migration and tube length of HBMECs were evaluated by wound healing and tube formation assays, respectively. The mRNA expression of APOA1 was determined by qRT-PCR. The proteins levels of APOA1, Bcl-2, Bax and Caspase-3 were determined by Western blot.


Introduction
Osteonecrosis of the femoral head (ONFH) most frequently comes from trauma or the use of corticosteroid and alcohol, while it is also related to blood dyscrasias and metabolic and coagulation disorders (1). Among the above factors, corticosteroid use is the most common cause of ONFH (2). Because treatment with glucocorticoids (one kind of corticosteroid) results in reduced blood ow and hypercoagulability, glucocorticoids damage the bone microvascular endothelial cells (BMECs) of the femoral head (3). BMECs form monolayer junctions attached to bone trabeculae, which are in direct contact with the ingredient in the blood to provide oxygen and nutrition for bone tissues and cells (4).
BMECs injury and dysfunction in femoral head are believed to play a signi cant role in the pathogenesis of femoral head necrosis (5). However, because of the complex etiology, the exact mechanisms of ONFH pathogenesis are not completely clear, and treatment with good long-term outcomes is yet to be developed.
The major apolipoproteins of plasma lipoproteins generally play important parts in preserving the structural integrity of lipoprotein particles and in physiological functions of lipoproteins (6).
Apolipoprotein A1 (APOA1), one type of apolipoprotein A family, is a primary protein component of highdensity lipoprotein (HDL) (7), and plays a role in reverse cholesterol transport that initiates multiple cellular pathways by binding to its receptor ABCA1 (8). Wu, X et al. (9) reported that down-expressed APOA1 improves risk prediction of Type-2 diabetes mellitus. Tuft Stavnes, H et al. reported that mRNA expression of APOA1 is a marker of longer survival in ovarian serous carcinoma effusions (10). However, the function of APOA1 in BMECs is still not clari ed yet.
Developed in ancient China, Traditional Chinese Medicine provides comprehensive holistic therapies and has been con rmed useful in the prevention and therapy of most pathologies worldwide (11).
Notoginseng, predominantly cultivated in China and Japan, is de ned in Chinese medicine as warm in nature (12). Panax notoginseng saponins (PNS) come from the active component of panax notoginseng root and can relieve swelling, promote blood clotting and alleviate pain with many biological activities such as immunomodulatory effects, antioxidation and anticancer properties (13). However, the effect of PNS on glucocorticoids-induced injury in human bone microvascular endothelial cells (HBMECs) remains to be elucidated.
In the present study, HBMECs were isolated from human femoral heads and used to investigate the effect of PNS on hydrocortisone (HC)-induced injury in HBMECs. To further elucidate the potential mechanism of this effect, we explore the role of APOA1 in the protective effect of PNS on HC-induced injury in HBMECs.

Materials And Methods
BMECs isolation, culture and identi cation Under aseptic condition, HBMECs were isolated from the femoral head samples and cultured with DMEM as described previously (14). Seven days after the culture, the morphology of the isolated HBMECs was observed by phase-contrast microscope. The expression levels of the markers CD31 (PA5-16301), CD133 (PA5-38014) and von Willebrand factor (vWF, PA5-16634) (Thermo Fisher Scienti c) were determined by immuno uorescence according to previous report (14,15).

Cell Treatment
HBMECs were cultured with 5% CO 2 at 37℃ with different concentrations (0.25, 0.5, 0.75, or 1.0 mg/ml) of HC (386698, Sigma) for 24 h to establish the HC-induced cell model (16). As shown in Fig. 2A cycles. Data were analyzed by the 2 −ΔΔCt method (17). The sequences of primers used in this research were displayed in Table 1.

Western Blot
HBMECs lysates were lysed for protein isolation and analyzed as previously described (16). In brief, HBMECs lysates were prepared with RIPA buffer (P0013, Beyotime, China) containing protease inhibitors. After protein containing compounds was centrifuged by high speed centrifuge (12,000 rpm, 5 min) at 4 °C, the supernatant was obtained. The total protein concentration was determined by double bicinchoninic Acid (BCA) assay kit (P0009, Beyotime, China). The protein was separated by 8% SDS-PAGE gel and transferred to a PVDF membrane (FFP24, Beyotime) at 4 °C for 2 h. Then, 5% skimmed milk was prepared using Tris-buffered saline with Tween-20 (TBS-T) to block the aspeci c antigen for 1 h. The membrane was washed 3 times with TBS-T and probed with primary antibody at 4℃ overnight. The speci c primary antibodies [APOA1, anti-apoptotic related-protein (Bcl-2), pro-apoptotic related-proteins (Bax, Caspase-3) and GAPDH] were listed in Table 2. The membrane was separated from the primary antibody and washed 3 times. The blots in membrane were probed with secondary antibody and washed 3 times. The secondary antibodies were HRP-conjugated secondary antibody (Goat Anti-Mouse, 1:2000, ab205719, Abcam, USA) and HRP-conjugated secondary antibody (Goat Anti-Rabbit, 1:2000, ab205718, Abcam, USA). BeyoECL Star (P0018FS, Beyotime) was used to visualize the target protein on the luminescent image analyzer (ImageQuant LAS4000 mini).

Scratch Wound Healing Assay
First, BMECs (5 × 10 5 /well) were cultured to 80-90% con uence in 6-well plates. After the medium was discarded, con uent HBMECs were scratched using a 10 µl tip, washed by serum-free DMEM, and incubated for 48 h. The evaluation of migratory activities was performed by counting migrating cells under a 100 × inverted microscope (Ts2r-FL, Nikon, Japan). Five random elds were chosen for each chamber. The following formula [(1 -the distance following healing/the distance prior to healing) × 100%] was used to calculated relative migration rate of HBMECs.

Tube Formation Assay
Matrigel was added to the pre-cooled 24-well plates at 150 µL/well. The plates were placed in an incubator at 37℃ with 5% CO 2 for 30 min to turn into glue for next experiments. After indicated treatment for 24 h, HBMECs were digested by trypsin and resuspended by serum-free DMEM. BMECs (5 × 10 5 /well) were planted into 24-well plates coated with matrigel. The HC (0.75 mg/mL) was added and the cells were cultured at 37℃ with 5% CO 2 and 95% humidity for 4 h. Next, the cells were photographed under a 100 × inverted phase contrast microscope. Three visual elds were randomly selected, and the tubule length was measured.

Statistical analysis
The data were shown as mean ± standard deviation (S.D.). Statistical signi cance in this study was analyzed by Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test. The analyses were performed by SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered statistically signi cant.

Results
HBMECs were identi ed and APOA1 participated in the protective effect of PNS on HC-inhibited cell viability The images of HBMECs were taken under a phase-contrast microscope (Fig. 1A). The cells reached 80% con uence with fusiform or polygonal shape, and exhibited a cobblestone-like morphology. As shown in Fig. 1B, the cells highly expressed CD31 and vWF, but did not express CD133, indicating that these cells were HBMECs.
We then investigated if HC-induced injury in HBMECs were related to the APOA1 expression. The viability of HC-injured HBMECs was determined by CCK-8 assay, and the APOA1 mRNA level was determined by qRT-PCR. As shown in Fig. 2A and B, after treatment with various doses of HC for 24 h, the cell viability was signi cantly reduced (P < 0.05 or P < 0.01 or P < 0.001) in parallel to a signi cant reduce in the mRNA level of APOA1 (P < 0.01 or P < 0.001). We detected the effect of PNS on cell viability and the mRNA level of APOA1 in HBMECs. As shown in Fig. 2C and D, after treatment with various doses of PNS for 24 h, the cell viability was not changed signi cantly while the mRNA level of APOA1 was signi cantly increased (P < 0.05 or P < 0.01 or P < 0.001). We explored whether PNS had a protective effect on HC-induced injury in HBMECs. As exhibited in Fig. 2E and F, PNS at the concentration range of 300-400 mg/ml signi cantly reversed part of the HC-induced cell injury (P < 0.05 or P < 0.01), and PNS at the concentration range of 200-400 mg/ml signi cantly increased the mRNA level of APOA1 (P < 0.05 or P < 0.001). Moreover, the effects of PNS on HC-induced injury in HBMECs and APOA1 mRNA level showed a dose-dependent manner; PNS (200-400 mg/ml) did not obviously change the viability of normal HBMECs, so we chose PNS (400 mg/ml) for next experiments. According to the above results, we speculated that APOA1 expression may be involved in the preventing effect of PNS on HC-induced injury in HBMECs.
APOA1 was involved in the preventing effect of PNS on HC-induced injury in HBMECs As shown in Fig. 3A and B, both protein and mRNA levels of APOA1 were signi cantly increased after APOA1 transfection (P < 0.001), while both of them were signi cantly decreased after siAPOA1 transfection (P < 0.001). We examined whether PNS and over-expressed APOA1 had a synergistic protective effect on HC-induced injury in HBMECs. As shown in Fig. 3C, relative to HC alone, the combination of HC and siAPOA1 further promoted cell injury (P < 0.001), but PNS treatment reversed this effect of siAPOA1 (P < 0.01). Relative to HC alone, the combination of PNS and HC treatment or APOA1 and HC treatment inhibited cell injury (P < 0.01); the effect of PNS was further enhanced by APOA1 treatment (P < 0.05), and the effect of APOA1 was further enhanced by PNS treatment (P < 0.05).

APOA1 was involved in the preventing effect of PNS on HC-induced apoptosis in HBMECs
The effects of APOA1, siAPOA1 and PNS treatment on HC-induced apoptosis in HBMECs were determined by ow cytometry and Western blot assays. According to Fig. 4A, relative to control group, HC led to a signi cantly enhanced apoptotic rate in HBMECs (P < 0.001) and the combination of HC and siAPOA1 further promoted the cell apoptotic rate (P < 0.001), while PNS treatment reversed this effect of siAPOA1 (P < 0.001). By contrast, PNS treatment or APOA1 treatment attenuated the HC-induced apoptosis (P < 0.001); the combination treatment of PNS and APOA1 further signi cantly attenuated the HC-induced apoptosis (P < 0.001); likewise, siAPOA1 treatment partially reversed this effect of PNS (P < 0.05). Cell injury is generally associated with changes in the expressions of apoptosis related-proteins.
According to Fig. 4B, compared with control group, HC obviously decreased the protein level of Bcl-2 and increased the protein levels of Bax and Caspase-3 in HBMECs; the combination of HC and siAPOA1 further decreased the protein level of Bcl-2 and increased the protein levels of Bax and Caspase-3; however, PNS treatment reversed this effect of siAPOA1. Conversely, PNS or APOA1 increased the protein level of Bcl-2 and decreased the protein levels of Bax and Caspase-3, and the combination treatment of PNS and APOA1 further signi cantly promoted the protein level of Bcl-2 and suppressed the protein levels of Bax and Caspase-3.

APOA1 was involved in the preventing effect of PNS on HC-inhibited migration and angiogenesis in
HBMECs According to Fig. 5A, compared with control group, HC signi cantly suppressed the migration rate in HBMECs (P < 0.001), and the combination of HC and siAPOA1 further suppressed the migration rate (P < 0.001); however, PNS treatment reversed this effect of siAPOA1 (P < 0.001). By contrast, PNS or APOA1 treatment promoted the migration rate (P < 0.001), and the combination treatment of PNS and APOA1 further signi cantly promoted the migration rate (P < 0.001); likewise, siAPOA1 treatment reversed this effect of PNS (P < 0.001).
According to Fig. 5B, compared with control group, HC signi cantly suppressed the tube length in HBMECs (P < 0.01) and the combination of HC and siAPOA1 further suppressed the tube length (P < 0.001); however, PNS treatment reversed this effect of siAPOA1 (P < 0.001). By contrast, PNS or APOA1 treatment promoted the tube length (P < 0.05), and the combination treatment of PNS and APOA1 further obviously promoted the tube length (P < 0.01); likewise, siAPOA1 treatment reversed this effect of PNS (P < 0.001). Thus, the data suggested that the changes of APOA1 expression by APOA1 or siAPOA1 transfection were involved in the preventing effect of PNS on HC-inhibited migration and angiogenesis in HBMECs.

Discussion
ONFH is still a disabling condition with ill-de ned etiology and pathogenesis (18). High levels of glucocorticoid-induced ONFH were observed in patients who underwent total hip replacement (19). We therefore used HC to injure HBMECs to establish a glucocorticoid-induced ONFH in vitro model and explored the protective role of PNS in HC-induced injury in HBMECs. This study may introduce PNS as a novel treatment strategy on ONFH.

Accumulated evidence indicates that PNS has protective effect on various injured cells. PNS exerts
protective effects against high glucose-induced oxidative injury in rat retinal capillary endothelial cells (13). PNS also attenuated the inhibitory effect of doxorubicin-induced damage on H9C2 cells (20). In this study, we found that PNS could attenuate the inhibitory effect of HC on the viability of HBMECs, implying PNS had a protective effect on HC-induced injury in HBMECs, which suggested that PNS had potential therapeutic effect on ONFH. More importantly, HC decreased the expression of APOA1 in a dosedependent manner, and PNS increased the expression of APOA1 in the same manner. We therefore suggested that the protective effect of PNS on HC-induced injury in HBMECs was related with the differential expression of APOA1. To further investigate the relationship between PNS and APOA1 in the protective effect on HC-induced cell injury, we transfected HBMECs with APOA1 or siAPOA1 to up-regulate or down-regulate APOA1 expression. The results revealed that PNS protected HBMECs against HCinduced injury through promoting the expression of APOA1. When the expression of APOA1 was suppressed, the protective effect of PNS on HC-injured HBMECs was also suppressed.
Previous studies have shown that apoptosis is induced by high concentration of glucocorticoids in osteocytes (21). Apoptosis is one type of programed cell death and plays a signi cant role in the loss of microvascular function (22). Apoptosis is modulated by many factors, especially Bcl-2 (inhibitors) and Bax (promoters), and by activation of the apoptotic cascade via caspase-3, the protein cleaved (activated) by which is considered as an executioner of apoptotic pathway and requires induction of apoptosis (23). In this study, we found that HC or APOA1 knockdown not only increased the apoptotic rate, but also inhibited the expression of Bcl-2 and promoted the expressions of Bax and Caspase-3. APOA1 knockdown further decreased HC-induced apoptosis; PNS inhibited apoptosis and reversed this effect of APOA1 knockdown; PNS and APOA1 over-expressions further signi cantly inhibited apoptosis. We demonstrated that PNS can protect HBMECs in HC condition by inhibiting apoptosis, and this effect of PNS was involved in the promotion of APOA1. Numerous studies have suggested PNS has antiapoptotic effect. For example, PNS attenuates cardiomyocyte apoptosis through mitochondrial pathway in natural aging rats (24). PNS protects against SH-SY5Y cell apoptosis induced by 6-hydroxydopamine (25) and K562 cell apoptosis (26). Besides, total PNS also exerts anti-apoptotic effect on rat bone marrow mesenchymal stem cells (27).
The migration of vascular endothelial cells is not only the key step of angiogenesis, but also the basis of neovascularization (28). Angiogenesis, the growth of new blood vessels from existing vessels, is an important aspect of the repair process of vascular injury (29). To further elucidate the role of PNS and APOA1 in HC-inhibited migration and tube formation of HBMECs, we co-cultured PNS with HBMECs exposed to HC condition. The results suggested that PNS treatment can promote the migration and tube formation of HBMECs through increasing the expression of APOA1, implying PNS promotes angiogenesis of HC-induced ONFH, and the elucidation of this mechanism may contribute to the treatment of ONFH. As for cancer, however, PNS inhibits migration in mouse breast carcinoma cell line (30); similar reports also showed that total PNS inhibits migration of human prostate cancer PC-3 cells (31). Additionally, apart from the effect on angiogenesis of HBMECs, PNS also promotes angiogenesis through AMPK and eNOS dependent pathways in HUVECs (32); PNS could enhance angiogenesis and the pro-angiogenic effects through the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways in vivo (HUVECs) and in vitro (zebra sh), respectively (33). Similar results reported that total PNS promotes angiogenesis deriving from rat bone marrow mesenchymal stem cells (34). By contrast, PNS suppresses angiogenesis of atherosclerotic plaque by decreasing vascular endothelial growth factors and the expression of nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (35).
Although our research illustrated the mechanism underlying PNS on cell viability, migration and tube formation in HC-injured HBMECs, more studies should be performed in appropriate animal models to further investigate clinical application of PNS on HC-injured ONFH. These attractive data revealed that the strategy that PNS promotes AOPA1 expression to protect HC-injured HBMECs might provide a promising therapeutic method for HC-injured ONFH.

Conclusions
Taken together, we demonstrated that PNS prevents HC-induced injury in HBMECs, and changes of APOA1 expression are crucial throughout the whole process. HC inhibits the APOA1 expression, while PNS promotes the APOA1 expression. PNS protect HC-injured HBMECs through promoting the expression of APOA1. Further, PNS and over-expressed APOA1 had a synergistic protective effect on HC-induced injury in HBMECs. Our results might provide evidence to support PNS use in the treatment of ONFH. No human or animals are involved in this research.

Availiability of Data and Materials
The analysed data sets generated during the study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable.

Availiability of Data and Materials
The analysed data sets generated during the study are available from the corresponding author on reasonable request.

Disclosure of Con ict-of-Interest
The authors declare no con icts of interest.    The effects of APOA1 expression on PNS preventing hydrocortisone (HC)-indured apoptosis in BMECs. HC+siAPOA1. The data were shown as mean ± standard deviation (S.D.).