Role of Bushen Huoxue Formula and transplanted endothelial progenitor cells play in promoting endplate microcirculation and attenuating intervertebral disc degeneration

Abstract

Objective

To explore whether Bushen Huoxue Formula (BSHXF) improves the angiogenesis ability of transplanted endothelial progenitor cells (EPCs) in endplate and its potential mechanism in delaying intervertebral disc degeneration (IDD).

Methods

BSHXF was analyzed via Ultra-High Performance Liquid Chromatography (UPLC). Rabbit axial compression lumbar IDD models were constructed and the effects of BSHXF, EPCs, and their combination in IDD were determined by MRI, histological evaluation, TUNEL, and immunofluorescence assays. Additionally, CCK-8 assay, flow cytometry, and tube formation assay were used to evaluate EPCs viability, proliferation, cell cycle and the angiogenesis ability of EPCs between groups.

Results

BSHXF and transplanted EPCs both attenuate the process of IDD in the rabbit model assessed by MRI, HE staining and Masson staining. TUNEL-positive NP cells were significantly reduced in the BSHXF group, EPCs group, and EPC + BSHXF group compared to the model group (P < 0.05), with the EPC + BSHXF group showing the most significant therapeutic effect. Immunofluorescence detection showed that VEGF, CD34 expression and quantity of microvessels in the endplate significantly increased in the EPC + BSHXF group compared to all the other groups (P < 0.05). Besides, the CCK-8 assay showed an upregulation of EPC viability and the tube formation assay demonstrated a significant increase in tube length and branching in EPCs cultured with BSHXF-containing serum (P < 0.05). Furthermore, BSHXF-containing serum increased VEGF expression in EPCs cultured in vitro (P < 0.05).

Conclusions

Both BSHXF and EPCs transplantation play an important role in increasing endplate angiogenesis and attenuating IDD. BSHXF can enhance the viability and tube-forming ability of EPCs and endplate microcirculation.

Abbreviations

BSHXF

Bushen Huoxue Formula

EPCs

endothelial progenitor cells

IDD

intervertebral disc degeneration

UPLC

Ultra-High Performance Liquid Chromatography

NP

nucleus pulposus

EP

cartilage endplate

AF

annulus fibrosus

ECM

extracellular matrix

FN

fibronectin

TCM

Traditional Chinese Medicine

1. Introduction

Intervertebral disc degeneration (IDD) is a pathophysiological process associated with various factors such as aging and has a profound impact on patients’ quality of life [1]. Intervertebral disc consists of three main structures: nucleus pulposus (NP), cartilage endplate (EP) and annulus fibrosus (AF). Among these, NP plays a crucial role in maintaining the balance of extracellular matrix (ECM). Apoptosis of NP cells, degradation of the ECM, and inflammation are considered to be main pathological manifestations of IDD [2,3]. It is worth noting that NP is the largest avascular tissue and mainly relies on nutrient diffusion from the EP. Reduced blood supply and inadequate nutrition of NP can lead to IDD [3]. Therefore, regulating blood supply and promoting angiogenesis in the EP are crucial for delaying the progression of IDD.

Vascular endothelial growth factor (VEGF) serves as a pivotal regulator in orchestrating vascular genesis and maintaining vascular homeostasis, crucial elements for preserving the microcirculation within the endplate and safeguarding intervertebral disc (IVD) health. Intervertebral disc degeneration (IDD) represents a prevalent musculoskeletal ailment characterized by structural and functional alterations in the intervertebral discs, often culminating in chronic low back pain and disability. Perturbed microcirculation within the endplate, stemming from diminished vascular genesis and vascular dysfunction, stands out as a pivotal contributor to IDD progression. VEGF, recognized as a potent inducer of vascular genesis, exerts profound impacts on endothelial cell proliferation, migration, and tube formation, thereby playing a pivotal role in upholding vascular integrity within the endplate microenvironment and fostering vascular genesis [4].

Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells primarily derived from bone marrow and play an important role in angiogenesis [5]. Transplantation of EPCs has been proved to have significant efficacy in treating different kinds of ischemic diseases [6,7]. However, there is currently a lack of research on the use of EPCs to improve endplate microcirculation and attenuate the process of IDD. If timely treatment can be performed during the ischemic stage of the vertebral endplate, improving local microcirculation, it may be possible to prevent or delay the occurrence of IDD.

Bushen Huoxue Formula (BSHXF) is a traditional Chinese decoction used to treat different kinds of diseases related to IDD. BSHXF was first recorded in the Great Achievement of Traumatology during the Qing Dynasty, where it was used to alleviate low back pain and promote blood circulation. Over the years, the Department of Spine Surgery at Guanghua Hospital has made slight modifications to the formula, and it is now widely utilized in patients with IDD, including postoperative and elderly patients. BSHXF has been clinically proven to be effective in treating IDD. In 2020, Feng [8] employed network pharmacology to study the mechanism of action of BSHXF in IDD. However, there remains a dearth of relevant experimental studies on this subject.

Therefore, we hypothesized that BSHXF could enhance the proliferation and angiogenesis of EPCs, thereby increasing the blood supply to the NP from the EP in IDD. For quality control and further research in the future, we first investigated BSHXF decoction and drug-containing serum by Ultra Performance Liquid Chromatography (UPLC). Then, a rabbit disc degeneration model was constructed and the impact of BSHXF in IDD was determined. Furthermore, we isolated EPCs from rabbit bone marrow and assessed the changes in proliferation and angiogenesis between the BSHXF treatment group and the normal group in an in vitro setting.

2. Methods

2.1. BSHXF solution and serum preparation

Thirteen herbs (Rehmanniae Radix 30g, Epimedii Folium 10g, Cuscuta chinensis Lam 10g, Eucommiae Cortex 10g, Lycii Fructus 10g, Corni Fructus 15g, Cistanches Herba 10g, Chuanxiong Rhizoma 15g, Carthami Flos 15g, Angelicae Pubescentis Radix 15g, Ginseng Radix et Rhizoma 15g, Astragali Radix 30g, Angelicae Sinensis Radix 15g) were purchased from Guanghua Hospital of Shanghai University of Traditional Chinese Medicine. The dosage for rabbits was 3.1 times that for humans [9]. For this study, a human requires 200g of herbs per 70 kg of body weight per day, thus a rabbit would need 2.86 g/kg of herbs.

All animal experiments were conducted in accordance with ethical guidelines and approved by the Laboratory Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. New Zealand rabbit (2-2.5 Kg, Shanghai JieSiJie Laboratory Animal Co., Ltd.) were gavaged with BSHXF (Rabbit BSHXF serum, n = 3) or distilled water (Rabbit con serum, n = 3) for 7 days. On day 7, disposable negative pressure blood collection vessel and blood collection needle was used to collect abdominal aortic blood 1h after gavage. Serum was extracted from the blood by centrifugation at 3000 rpm for 15 min. The rabbit BSHXF serum and rabbit control serum were harvest separately and then sterilized in a 55 °C water bath for 30 min and stored at −20 °C [10].

2.2. UPLC analysis

Ingredients of BSHXF were identified by UPLC which was also used to control its quality. Instrument: Waters ACQUITY UPLC H Class-Waters Synapt G2 Si QTOF. Analysis method of the chromatographic profile: Stationary phase: Waters Acquity HSS T3, (100 × 2.1 mm, 1.8 μm); mobile phase: aqueous with 0.1% formic acid (I) and acetonitrile with 0.1% formic acid (II) (0–15 min, 2–30% II; 15–20 min, 30–50% II; 20–25 min, 50–95% II; 25–29 min, 95% II; 27–30 min, 2% II) [11,12].

2.3. EPCs isolation and culture

Rabbit femur bone marrow was collected under sterile conditions and then transferred into a test tube with an equal volume of sterile PBS solution. The mixture was centrifuged, and the supernatant was replaced with PBS solution and thoroughly mixed. The bone marrow cell suspension was then layered onto a tube containing Ficoll solution, followed by centrifugation to separate the mononuclear cell layer. The mononuclear cells were mixed with PBS solution after centrifugation. Isolated mononuclear cells were placed in an endothelial progenitor cell-specific medium at a density of 1 × 106-1 × 107/cm2 in a precoated dish with fibronectin (FN). The cells were incubated at 37 °C with 5% CO2 and saturated humidity for 2 h. The suspension was removed, and the cells were centrifuged again (4 °C, 300×g for 6 min). The supernatant was discarded, and then the endothelial progenitor cell-specific culture medium was added and mixed for secondary inoculation in the culture flask/dish precoated with FN and recorded as P0. After 7 days, when the adherent cells spread all over the bottom wall of the culture dish, the cells were prepared for digestion and passaging. The unpatched cells were washed twice with PBS and digested with 0.05% trypsin-0.02% EDTA, and then seeded in new dishes at a density of 5 × 106/(ml-dish) for passaging, which was recorded as the P1 generation. When cells were passed to the P3 generation, cell identification was performed [13].

3. Identification of EPCs

The morphology of adherent cells was observed under an inverted aberration microscope. The digested cultured P3 generation cells were centrifuged, and the supernatant was removed. Then, 5 ml of culture medium and 12.5 pg (density 2.5 pg/ml) of DiL-ac-LDL (red) were added and incubated at 37 °C for 1 h. The cells were fixed with 2% paraformaldehyde for 10 min and washed twice with PBS. Next, 50 μg (density 10 μg/ml) of FITC-UEA-1 (green) was added for 1 h at 37 °C, and the cells were observed using laser confocal microscopy. Cells positive for both DiL-ac-LDL and FITC-UEA-1 double-staining (appearing yellow) were considered to be differentiating EPCs [14].

3.1. Animal modeling and grouping

New Zealand rabbits (2–2.5 kg, Shanghai JieSiJie Laboratory Animal Co., Ltd.) were raised with abundant food and water, maintaining a temperature of 23 ± 2 °C and a 12-h light/dark cycle.

After one week of adaptive feeding, the rabbits were randomly divided into the IDD model group (n = 24) and the control group (n = 6). Rabbits in the model group received a lumbar disc degeneration model establishment through an exernal compressive device [15]. Sodium pentobarbital was injected intraperitoneally (30 mg/kg) to induce anesthesia in rabbits before the surgery was performed through a dorsal approach from the level of L4 vertebral body to L5. Muscles on both sides of the spinous process were bluntly separated and then the lumbar vertebral bodies were exposed. The puncture point of the vertebral body was identified 2 mm above the caudal end of the junction between the transverse process and the vertebral body. The average weight of the custom-made external loading device was 50g. The device was placed externally and attached to 2 K-wires (diameter, 2.0 mm) inserted into the vertebral bodies of L4 and L5 by the use of a variable-speed electric drill. After confirming the absence of active bleeding, spinal cord injury and abdominal cavity injury, the wound was closed and axial compression stress of 2.4 MPa was applied to the lumbar spine. Rabbits in the control group underwent surgical placement of the 2 K-wires with the external device attached but no compression applied. The wounds on the skin were disinfected daily with iodophor. Additionally, gentamicin 40,000 U intramuscular injections were administered daily for the first three days after the operation to prevent infection and the device was measured and adjusted every day to maintain a constant axial stress. The wounds on the skin were disinfected daily with iodophor until they healed.

4 weeks later, the IDD model group was further divided into four randomly assigned groups: 1) IDD rabbits treated with transplantation of EPCs in the endplate, 2) IDD rabbits treated with BSHXF, 3) IDD rabbits treated with BSHXF and transplantation of EPCs in the endplate, 4) IDD rabbits with no treatment. Each group consisted of six rabbits (n = 6). This study wasd blinded and the scientists who conduct the experiment did not know the condition of grouping no matter in the animal experiment or in vitro experiment.

3.2. BSHXF gavage and EPCs transplantation

Prepared BSHXF solution was administered to the rabbits via gastric gavage using a 20 ml syringe with a gavage needle. The process was conducted gently and steadily to avoid injuring the animals' oral cavity and esophagus. BSHXF solution was administered once daily for 28 days.

After anesthetizing the rabbits, they were placed in a prone position on the operating table, and use a 1 ml syringe to draw up the EPCs suspension for injection beneath the vertebral endplate. The injection site is selected between the L4 and L5 vertebrae, approximately 1 cm lateral to the midline and at an angle of approximately 60° from the horizontal line. Upon insertion of the needle into the vertebra, a firm resistance was felt. Confirm the position of the needle tip using the anterior view (Fig. 1A) and the lateral view (Fig. 1B) of X-ray. Once the needle tip reached the endplate, the EPC suspension was slowly injected over a duration of 1 min.

Fig. 1.

X-ray guided EPCs transplantation puncture point. A shows the anterior view, B shows the lateral view.

3.3. Magnetic resonance evaluation

The radiological evaluation of IDD was conducted using a Siemens Trio Tim 3.0T MR scanner (Siemens Medical Solutions, Erlangen, Germany).Throughout the MRI examination, all rabbits were anesthetized. The animals were placed prone in the MR scanner, and the body temperature was maintained at 37 °C using circulating heated air. A serial T2-weighted sagittal plane covering the entire experimental disc area was obtained using the following parameter settings: repetition time, 2800 ms; echo time, 120 ms; field of view, 260; slice thickness, 2 mm. T2-weighted sagittal images of each targeted disc was collected for the grade evaluation. Two radiologists assessed the disc degeneration levels in accordance with the standard recommended by Pfirrmann et al. [16].

3.4. Histological evaluation

After magnetic resonance imaging, the experimental animals were euthanized under excessive anesthesia. The intervertebral disc tissues were isolated and fixed in 4% paraformaldehyde for 24 h. Decalcification of the tissues was performed using EDTA decalcification solution, with regular solution changes. The dehydrated tissues were embedded in paraffin, sectioned, and subjected to various staining techniques including hematoxylin and eosin, Masson's trichrome, and aniline blue. Pathological evaluations of the intervertebral disc NP, AF and ECM were conducted using the Mesuda grading system [17].

3.5. TUNEL assay

The prepared paraffin sections were deparaffinized by placing them vertically in a glass container and incubating them in a 60 °C oven for 1 h until the paraffin melted. Subsequently, the sections were transferred to a fume hood and immersed in xylene. Deparaffinization and hydration were then carried out by sequential immersion in xylene I and xylene II for 10 min each, followed by ethanol washes of decreasing concentrations (anhydrous ethanol, 95%, 80%, and 75%) for 5 min each. The sections were washed three times with PBS for 5 min each. For labeling and detection, the sections underwent various steps involving Proteinase K treatment, equilibration, labeling, and incubation. Finally, the sections were counterstained with DAPI and mounted using an anti-fluorescence quenching mounting medium for observation under a confocal microscope.

To analyze the results, Image J software was utilized. DAPI-stained cells were counted by converting the images to 8-bit format, applying Gaussian blur, setting pixel intensity threshold, and performing watershed segmentation. The cell count was recorded. The same procedure was applied to count TUNEL-positive cells. The percentage of apoptotic cells in NP was calculated by dividing the count of TUNEL-positive cells by the count of DAPI-stained cells, multiplied by 100%.

3.5.1. Immunofluorescence assay

Tissue sections were processed for deparaffinization, washing, fixation, permeabilization, and blocking. Primary antibodies against VEGF and CD34 were applied without washing and incubated overnight at 4 °C. After incubation, the sections were washed with PBS, followed by incubation with fluorescent secondary antibodies for 1 h at 37 °C. Since it was a double staining, VEGF antibody and CD34 antibody needed to be from different species. The sections were washed with PBS three times for 5 min each. Fluorescent secondary antibodies were added and incubated at room temperature in the dark for 1 h. After washing the sections with PBS in the dark for 5 min three times, DAPI staining was performed at room temperature for 30 min, followed by a 5-min wash in the dark. The sections were then mounted using an anti-fluorescence quenching mounting medium and photographed. The fluorescent images were observed using Slide Viewer software. The viewing distance was adjusted to a depth of 500 μm to observe the entire intervertebral disc tissue and the microvessels distributed in the endplate. VEGF expression was detected with red fluorescence, CD34 expression with green fluorescence, and DAPI staining with blue fluorescence. The triple fluorescence within the endplate indicated the microvessels and was counted by zooming in on the images.

3.5.2. CCK-8 assay

CCK-8 assay was performed to evaluate the viability of EPCs. Cells were seeded at a density of 1.5 × 104 cells/well in 96-well plates. EPCs were treated with rabbit BSHXF-containing serum for 0, 12, 24, 36, and 48 h to assess the effect of BSHXF on cell viability. Afterwards, 10 μl of CCK8 reagent and 90 μl of culture media were added to each well and incubated for 2 h at 37 °C. The absorbance values at 450 nm (OD450) were measured using an enzyme marker, with four replicate wells for each group.

3.5.3. Flow cytometry assay

The proliferation level of EPCs was assessed through cell cycle assays using flow cytometry. Forty-eight hours after drug administration, EPCs from each group were analyzed. The EPC cycle was divided into G1/G0, S, and G2/M phases, and the proliferation index of cells was estimated based on the cell cycle. The calculation formula used was (S + G2M)/(G1+S + G2M) × 100%. The proliferation index provides an indication of the proliferative activity of cells to a certain extent.

3.5.4. Tube formation assay

Matrigel matrix was evenly coated onto a 48-well plate. EPCs were cultured in regular medium or BSHXF serum-containing medium with drugs for 24 h. After removing the culture medium, the cells were digested and prepared into a uniform EPC suspension. Subsequently, an equal volume of the suspension was seeded onto the 48-well plate. After incubating for 8 h, inverted microscopy was used to observe the formation of tube-like structures. The angiogenesis network was quantified using the Angiogenesis Analyzer plugin in ImageJ, analyzing the changes in total vessel length and vessel branching numbers.

3.6. Western blot

EPCs were lysed with RIPA buffer to extract total EPC protein. The total protein concentration was measured using the BCA protein kit (Pierce, Rockford, IL, USA). The protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then blocked in TBST with 5% skim milk at room temperature for 1 h and subsequently incubated overnight at 4 °C with primary antibodies, including rabbit anti-VEGF (1:1000, Bioss), rabbit anti-CD34 (1:1000, Bioss), and rabbit anti-GAPDH (1:4000, Abcam). After washing with TBST, the membranes were incubated with secondary antibodies at room temperature for 1 h. Protein bands were visualized using an ECL reagent.

3.7. Statistical analysis

One-way ANOVA and Bonferroni test were used for multiple comparison and t-test were performed to compare two groups if the data followed a normal distribution and homogeneous variances. For ordinal data, Kruskal-Wallis test was used for multiple group comparisons and Mann-Whitney U test was used to compare two groups. P < 0.05 was considered significant. Data analysis was performed with SPSS version 24.0 and GraphPad Prism version 8.0.

4. Results

4.1. Analysis of BSHXF chemical components

BSHXF solution was investigated by UPLC for quality control and further research in the future. A total of 10 peaks were identified in BSHXF solution and the most qualitative compounds in BSHXF solution include hydroxysafflor yellow A, loganin, pinoresinol diglucoside, echinacoside, ferulic acid, calycosin-7-O-β-D-glucoside, acteoside, icariin, astragaloside A and columbianadin. The chromatogram of BSHXF solution is shown in Fig. 2A and qualitative compounds of BSHXF solution were summarized in Fig. 2B. Besides, BSHXF-containing serum from rabbits were also investigated by UPLC and few peaks were observed in the chromatogram (Fig. 2C).

Fig. 2.

A. UPLC analysis of ingredients from the BSHXF solution. neg: Negative Total-ion chromatograms of BSHXF; pos: Positive Total-ion chromatograms of BSHXF. B. Chemical components of BSHXF solution. C. UPLC analysis of ingredients from the BSHXF-containing serum. Blood neg: Negative Total-ion chromatograms of drug-containing serum. Blood pos: Positive Total-ion chromatograms drug-containing serum.

4.2. EPCs identification

After cell separation, the cells appeared oval-shaped under the microscope. 1 day later, the cells begin to adhere to the substrate. After 3 days, some adherent cells exhibit spindle-shaped morphology. Cell colonies start to emerge after 7 days of inoculation, with cells tightly packed and displaying clustered growth (Fig. 3A–F). Under fluorescence microscopy, EPCs that have engulfed Dil-ac-LDL exhibit red fluorescence, while EPCs that have membrane-bound FITC-UEA-1 display green fluorescence. Upon merging of the two fluorescence signals, a yellow fluorescence is observed, indicating the presence of EPCs characteristics. This confirms that the isolated cells were indeed EPCs (Fig. 4A–C).

Fig. 3.

Observation of EPCs derived from rabbit bone marrow under a light microscope. A ( × 100) and B ( × 200) represent freshly isolated cells, which exhibited an oval shape. C ( × 100) and D ( × 200) represent cells after 3 days of cultivation, where some EPCs adhered to the culture dish and displayed a spindle or elongated shape. E ( × 100) and F ( × 200) depict the morphology of cells after 7 days of cultivation, with cells adhering to the dish, exhibiting clustered growth, and arranging closely together.

Fig. 4.

EPCs engulfment experiment. A shows EPCs exhibiting red fluorescence after engulfing Dil-ac-LDL. B exhibits green fluorescence after binding with FITC-UEA-1. C displays yellow fluorescence after fusion.

5. BSHXF and EPCs transplantation decelerate the process of IDD

5.1. MRI evaluation

Representative T2-weighted sagittal MR images illustrating the changes in signal intensity and the structural characteristics of lumbar discs in different groups were shown in Fig. 5A–J. In the control group, the intervertebral disc structure appeared nearly intact and homogeneous, exhibiting a high signal intensity. The differentiation between NP and AF was clearly visible. The grade of the control group is 1.17 ± 0.39, which is significantly lower than all the other 4 groups(p < 0.05). In the model group, a reduction in disc height was observed, accompanied by a loss of distinction between NP and AF and a lower signal intensity. Both the EPC group and the BSHXF group exhibited a slightly brighter T2 signal intensity compared to the model group, although the difference was not statistically significant. In the EPC + BSHXF group, the differentiation between NP and AF was unclear, showing an intermediate decrease in signal intensity, and a slight decrease in disc height, which is significantly lower than the model group (3.08 ± 0.29 vs.4.33 ± 0.65, p < 0.05). Fig. 5K shows the results of MRI grades according to Pfirrmann's classification.

Fig. 5.

Representative MR images of different groups and their grades of Pfirrmann's classification. A, B: Control group; C, D: Model group; E, F: EPCs transplantation group; G, H: BSHXF group; I, J: EPC + BSHXF group. K shows the results of MRI grades according to Pfirrmann's classification.

5.2. Histological evaluation

Representative images of hematoxylin and eosin staining and Masson staining in the 5 groups are showed in Fig. 6A-6O and Fig. 7A-7O. Fig. 6P shows then results of histological scores according to Masuda evaluation. In the control group, the histological morphology appeared nearly intact, with a U-shaped AF structure projecting slightly to both sides without rupture. The EP cells exhibited normal morphology, and the EP thickness was within the normal range. The NP contained a gelatinous matrix and large vacuoles. Masson staining is employed to provide a better visualization of intervertebral disc tissue, especially the collagen content in the NP. In the control group, the collagen distribution within the NP tissue is uniform, with clear boundaries and no signs of compression. The grade of control group is 5.17 ± 0.39, which is significantly lower than the other 4 groups(p < 0.05). In the model group, extensive fiber rupture and snake-like structures were observed in the AF. The EP thickness was significantly reduced, and the number of EP cells decreased significantly. Moreover, a severe disruption occurred between the AF and NP. Additionally, the number of NP cells decreased significantly, and the ECM showed moderate to severe condensation. Masson staining of the model group demonstrates a severe loss of collagen within the NP, accompanied by significant consolidation.

Fig. 6.

HE staining of intervertebral discs in all groups and their scores according to Masuda evaluation. A, B, C: Control group; D, E, F: Model group; G, H, I: EPCs transplantation group; J, K, L: BSHXF group; M, N, O: EPC + BSHXF group. A, D, G, J, M: intervertebral discs of different groups (*50); B, E, H, K, N: NP tissue of different groups (*200); C, F, I, L, O: AF tissue of different groups. K: Results of histological scores according to Masuda evaluation.

Fig. 7.

Masson staining of intervertebral discs in all groups: A, B, C: Control group; D, E, F: Model group; G, H, I: EPCs transplantation group; J, K, L: BSHXF group; M, N, O: EPC + BSHXF group. A,D,G,J,M: intervertebral discs of different groups(*50); B,E,H,K,N: NP tissue of different groups(*200); C,F,I,L,O: AF tissue of different groups.

In the EPC transplantation group, compared to the model group, there was a reduction in the severity of AF rupture. The structure of AF can be identified, but there are still a significant number of snake-like fibers present. The boundary between the AF and the NP is clearer compared to the model group. The NP tissue appears fragmented or island-like and there is a significant reduction in the number of NP cells, and the ECM shows mild consolidation. Masson staining confirms the severe loss of collagen within the NP with mild consolidation. However, the grade shows no significant difference with the model group.

In BSHXF group, compared to the model group, the integrity of the AF is higher, with fewer snake-like fibers and better continuity. The boundary between AF and NP is clearer, although the penetration of annular fibers into the nucleus can still be observed. The NP tissue in this group shows less pronounced fragmented or island-like structures and has a higher level of uniformity. The loss of NP cells is milder compared to the model and EPC transplantation groups, and the ECM shows mild consolidation. Masson staining indicates better preservation of collagen within NP. The grade of this group is significantly lower than the model group(8.50 ± 1.31 vs. 11.67 ± 1.30, p < 0.05).

In EPC + BSHXF group, the integrity of AF and NP tissue is higher compared to other intervention groups. The annular fibers still show signs of compression but are not disarranged. The NP exhibits a high level of uniformity with minimal fragmented or island-like distribution, and there are only very few gaps within the NP tissue. The loss of NP cells is minimal, and the ECM shows only mild consolidation. The grade of this group was significantly lower than the model group(8.08 ± 1.08 vs. 11.67 ± 1.30, p < 0.05).

5.3. NP cells apoptosis evaluation

TUNEL experiments were conducted to detect the proportion of apoptotic NP cells in each group(Fig. 8A–K). In the control group, the proportion of TUNEL-positive cells in the NP tissue is low, indicating that only a small number of NP cells undergo apoptosis in physiological situations. In the model group, the proportion of TUNEL-positive cells is 71.2 ± 6.71%, which is significantly increased than the control group(p < 0.05). In the EPC transplantation group, the proportion of TUNEL-positive cells is significantly increased compared to the control group (49.93 ± 5.38 vs. 25.19 ± 4.66, p < 0.05) and deceased than the model group(p < 0.05). In BSHXF group, the proportion of TUNEL-positive cells is 22.37 ± 5.02, which is furtherly reduced compared to the EPC transplantation group and model group, suggesting a stronger ability to diminish NP cell apoptosis, and the difference between BSHXF and the control group is not statistically significant. In the EPC + BSHXF group, the proportion of TUNEL-positive cells reaches its lowest level, 19.86 ± 3.30, which is also lower than the EPCs transplantation group and the model group(p < 0.05).

Fig. 8.

Representative immunofluorescence images of TUNEL staining in rabbit discs and the quantification of the TUNEL positive signals of different groups.

A, B: Control group; C, D: Model group; E, F: EPCs transplantation group; G, H: BSHXF group; I, J: EPC + BSHXF group. K: Results of TUNEL positive cells of different groups.

5.4. BSHXF and EPCs transplantation promote angiogenesis in endplate

Immunofluorescence staining revealed that in the control group, the distribution of microvessels in the endplate was uniform, with a higher number of lumens, indicating normal blood circulation capable of meeting the nutritional demands of NP tissue. In the model group, the [18] number of microvessels was significantly reduced compared to the model group(36.90 ± 13.12 vs. 209.6 ± 27.46, p < 0.01), with only a small number of unevenly distributed microvessels remaining and the blood circulation in the endplate is severely impaired. In BSHXF group, the number of microvessels showed no significant difference with the EPC transplantation group(118.3 ± 34.78 vs. 135.0 ± 29.18, p > 0.05) and the distribution remains uneven, but the quantity of the lumens in these two groups is less than the control group and more than the model group(p < 0.05). In the EPC + BSHXF group, the number of blood vessels is 175.4 ± 26.36, which is more than that of BSHXF group and EPCs transplantation group(p < 0.05). The lumens are evenly distributed, and the blood circulation further improves(Fig. 9A–F).

Fig. 9.

Microvessels in endplate of discs in different groups (500 μm). A: Control group, B: Model group, C: EPCs transplantation group, D: BSHXF group, E BSHXF + EPC group, F: Statistical result of microvessel quantity in endplate of different groups.

To investigate the impact of BSHXF and EPCs transplantation on angiogenesis in EP in the process of IDD, immunofluorescence staining for VEGF and CD34 was performed. Our findings demonstrate that BSHXF enhances the expression of VEGF and CD34, leading to the formation of a greater number of microvessels. Notably, the combined use of BSHXF and EPCs transplantation exhibits even more pronounced effects (Fig. 10). These results strongly suggest that BSHXF exerts a positive regulatory effect on endplate angiogenesis by promoting the activity of transplanted EPCs.

Fig. 10.

Representative images of angiogenesis in endplate in different groups (50 μm). Red fluorescence represents VEGF, Green fluorescence represents CD34, Blue fluorescence represents DAPI, merge shows the fusion of the three fluorescent stains, indicating the angiogenesis in endplate.

5.5. BSHXF-containing serum upregulates cell viability of EPCs in vitro

Considering the crucial role of BSHXF in endplate angiogenesis and IDD in vivo, we explored whether BSHXF exhibits protective effects on EPCs in vitro. A CCK-8 assay was conducted to assess the viability of EPCs. The results showed that BSHXF-containing serum had a positive regulatory effect on EPC viability, as demonstrated by increased cell viability compared to the control group after 24h(Fig. 11A). However, there were no significant differences observed in cell cycle analysis or the proliferative activity of EPCs between BSHXF group and the control group, as estimated by the calculation formula ((S + G2M)/(G1+S + G2M) × 100%) (Fig. 11B–C).

Fig. 11.

EPCs viability and cell cycle detection. A: Cell viability of BSHXF-containing serum group and the control group was detected by CCK-8 assay at different time; B: Cell cycle and proliferation of EPCs in the control group detected by flow cytometry. C: Cell cycle and proliferation of EPCs in BSHXF-containing serum group.

BSHXF-containing serum improves EPCs tube formation and VEGF expression in vitro.

A tube formation assay was conduct to evaluate the ability of EPCs to form tubes, indicative of their angiogenic potential. EPCs cultured in BSHXF-containing serum for 24 h exhibited enhanced tube formation capability. Analysis of the blood vessel network using Angiogenesis Analyzer revealed that the total length and branching number of the tubes formed by the EPCs in the BSHXF group were significantly greater than those in the control group, with statistical significance (P < 0.05) (Fig. 12A–D). Furthermore, the expression of vascular endothelial growth factor (VEGF) was assessed in EPCs. The results revealed a significant upregulation of VEGF expression in EPCs cultured with BSHXF-containing serum compared to the control group (P < 0.01). This suggests that BSHXF treatment promotes VEGF expression in EPCs, which is an important factor in angiogenesis (Fig. 12E). These findings collectively indicate that BSHXF enhances cell viability, tube formation ability, and VEGF expression in EPCs, highlighting its potential protective role in endplate angiogenesis and IDD.

Fig. 12.

EPCs tube formation ability and VEGF expression detection. A: Representative picture of EPCs cultured for 24 h in normal condition. B: Representative picture of EPCs cultured for 24 h with BSHXF-containing serum. C: Total length of blood vessel network of the two groups, as measured by Angiogenesis Analyzer. D: The branching number of the blood vessel network of the two groups. E: Western blot of VEGF expression of the two groups.

6. Discussion

Traditional Chinese Medicine (TCM) boasts a rich history spanning thousands of years and has developed a comprehensive system of theories, diagnostics, and therapies, particularly in Asian countries, notably China. It plays a significant role in the field of complementary and alternative medicine [19]. According to TCM theory, IDD and its associated diseases are primarily attributed to deficiency. For instance, low back pain is known to be caused by "Shen (Kidney) deficiency," and it is believed that "all pain is caused by qi and blood stasis." Based on the pathophysiology of IDD and the theory of Shen deficiency and blood stasis, BSHXF was developed to tonigy Shen and promote blood circulation. The formulation of BSHXF used in this study was based on the Qing'e Pill described in the Formulary of the Bureau of Taiping People's Welfare Pharmacy (Tai Ping Hui Min He Ji Ju Fang).

Several studies have demonstrated the favorable efficacy of BSHXF in reducing symptoms and improving function in patients with IDD [20]. Furthermore, laboratory investigations have revealed that BSHXF-containing serum effectively attenuate the loss of proteoglycan and type II collagen in degenerating discs, reduces endplate calcification and delays the progression of IDD [21,22]. These findings collectively support the potential of BSHXF as a viable therapeutic option for the management and prevention of IDD-related diseases.

However, most previous studies have focused on the research of NP cells, while the theories of TCM on promoting blood circulation and removing blood stasis share similarities with improving endplate microcirculation in IDD. Existing studies have shown that some TCM formulas have a promoting effect on the proliferation, migration, adhesion, and angiogenesis of EPCs [23,24]. Therefore, this study is based on the nutritional supply of intervertebral discs and aims to explore the effects of the BSHXF on EPCs from the perspective of endplate microcirculation.

EPCs are precursor cells of endothelial cells that play an important role in angiogenesis. Asahara et al. [25] discovered that EPCs can be mobilized from the bone marrow and enter circulation to exert their effects. Doyle et al. [26] proposed that EPCs can participate in endothelial differentiation and contribute to angiogenesis. Therefore, researchers have been exploring the use of EPCs transplantation for the treatment of ischemic diseases. It is generally recognized that EPCs possess the biological characteristics of simultaneous positivity for CD34, CD133, and VEGFR2 (KDR) [27]. Furthermore, the uptake of Dil-ac-LDL and FITC-UEA-1 confirms the differentiation of EPCs, and their identification can be achieved through morphological analysis or the detection of Dil-ac-LDL and FITC-UEA-1 uptake [28], which is generally sufficient for EPC identification.

The transplantation methods for EPCs have been studied and applied in various ischemic diseases, but a unified approach is still lacking. Currently, the main transplantation routes are local injection and intravenous injection via peripheral blood. Some studies suggest that intravenous injection is convenient, but EPCs administered via peripheral blood may be influenced by systemic conditions and homing to different sites, making it difficult to control the local concentration of EPCs. Additionally, EPCs may form clots or induce rejection when present in peripheral blood. On the other hand, local transplantation can ensure a relatively stable concentration of EPCs at the ischemic site, avoiding the influence of other factors. However, its disadvantage lies in the complexity of the procedure, particularly for targeting deep organs or tissues. This study focuses on the blood supply of the endplate, where local transplantation provides greater stability and controllability. Furthermore, the use of an X-ray-guided injector needle helps avoid the possibility of inaccurate positioning in deep tissue during local transplantation. In terms of the number of transplanted EPCs, different studies have targeted different sites and selected varying cell quantities. Numerous studies have indicated that locally transplanted EPCs are generally in the range of 1 × 105 to 1 × 10^6. MIFUJI et al. [29] performed local transplantation of EPCs for the treatment of fractures, using a quantity of 2 × 10⁵ EPCs. Correa A et al. [30] injected 6.6 × 10⁵ EPCs derived from umbilical cord blood into the myocardium for the treatment of myocardial infarction. Currently, there are no reports on the quantity of EPCs transplantation in endplate. Considering the species and volume differences in different tissues, a relatively small quantity is required for injection. This study selected a quantity of 1 × 10⁵ EPCs suspended in 1 ml of PBS to control the injection time within 1 min for the endplate site injection.

Regarding the selection of animal models, there are various types for IDD, including compression models, upright models and needle puncture models [[31], [32], [33]]. Due to the diverse causes of IDD, it is necessary to select a model where endplate circulation reduction and IDD occur simultaneously. The compression model not only induces IDD stably but also significantly impacts endplate circulation. New Zealand rabbits are easy to handle, moderately priced, and suitable for this study's animal model. Additionally, this study evaluated the degree of IDD using the classic MRI Pfirrmann grading and Masuda scoring in histology. This confirms the successful establishment of the animal model for IDD and verifies the effectiveness of BSHXF and EPCs transplantation in slowing the process of IDD. The axial compression model notably decreased the continuity of AF and blurred the boundary with the NP. Besides, there was a significant decrease in the number of NP cells and ECM loss. However, the administration of BSHXF and EPC transplantation could delay these pathological changes to varying degrees. BSHXF exhibited a stronger ability to delay pathological changes, particularly in the protection of NP cells and the ECM. The AF of the BSHXF group had higher continuity and homogeneity. When BSHXF administration was combined with EPC transplantation, this protective effect may be further enhanced, leading to a further reduction in the loss of ECM.

CD34 is a cell surface antigen widely expressed on human and other mammalian hematopoietic stem/progenitor cells or mature vascular endothelial cells [34]. In this study, VEGF and CD34 were fluorescently double-stained to label the endplate microvasculature, and the lumens were counted to observe the effects of different interventions on endplate blood circulation [35]. The results showed that after modeling, endplate circulation was severely impaired. However, EPCs transplantation and BSHXF administration could partially restore endplate circulation, with a more significant effect observed when the two interventions were combined. This result is consistent with the degree of IDD in vivo, indicating a close correlation between IDD and endplate circulation. Furthermore, EPCs endplate transplantation may have been involved in local angiogenesis, improving endplate blood supply. In addition, CCK-8 indicated that BSHXF-containing serum enhanced the viability of EPCs, with a significant difference observed at 24 h. Flow cytometry analysis of the cell cycle showed that BSHXF had no effect on the cell cycle and proliferation of EPCs, suggesting that BSHXF may enhance the cellular activity of EPCs by reducing apoptosis. Tube formation experiments and Western blot results indicated that BSHXF treatment significantly enhanced the tube formation ability of EPCs and promoted the expression of VEGF, thereby increasing their angiogenic capacity.

VEGF-mediated vascular genesis is a fundamental process in restoring and maintaining endplate microcirculation, crucial for nutrient and oxygen supply to the avascular intervertebral disc. In the context of IDD, dysregulated VEGF signaling leads to vascular dysfunction, resulting in hypoxia, nutrient deprivation, and accumulation of metabolic waste products within the disc, exacerbating degenerative processes. Downregulation of VEGF expression in degenerated endplates perpetuates vascular dysfunction, establishing a vicious cycle of disc degeneration. Bushen Huoxue Formula (BSHXF) and transplanted endothelial progenitor cells (EPCs) represent promising therapeutic strategies for restoring endplate microcirculation and ameliorating IDD progression. The pharmacological action of BSHXF involves upregulating VEGF expression, promoting vascular genesis within the endplate region, thereby improving nutrient delivery and metabolic exchange within the intervertebral disc. Similarly, transplantation of EPCs increases endogenous VEGF production, facilitating neovascularization, fostering a microenvironment conducive to disc regeneration and repair.

All the results above verified that BSHXF attenuates IDD through promotes angiogenesis of transplanted EPCs in endplate. BSHXF contains 13 botanical drugs, and the main components of BSHXF and their molecular information are shown in Fig. 2B. Hydroxysafflor yellow A, the main ingredient of BSHXF, was shown to promote angiogenesis via varied pathways [36,37]. Yung-Hsin Cheng et al. found that ferulic acid-gelatin can treat IDD from the damage caused by oxidative stress [38]. Icariin was also proven to have anti-apoptosis and anti-inflammatory effects and regulate stem cell migration in IDD [[39], [40], [41]]. Du et al. demonstrated that astragaloside can alleviate damage in NP cells via inhibit miR-223/JAK2/STAT1 Pathway [42].

There are some limitations in this study. First, IDD is a complex process that involves biomechanics and a series of pathological and physiological changes. Axial compression model may not fully simulate the degenerative process. Further improvements in the modeling approach are needed in the future. Second, the timing and frequency of EPC transplantation require further investigation to determine whether transplantation at different time points or altering transplantation frequency can achieve better therapeutic effects without causing tissue damage. In addition, further research is necessary to elucidate the molecular mechanisms of action of different BSHXF ingredients in attenuating IDD, providing a basis for understanding their therapeutic effects.

7. Conclusion

Both BSHXF gavage and local transplantation of EPCs demonstrate the ability to enhance endplate microcirculation and mitigate the progression of IDD. When both intervention methods are combined, the efficacy of improvement is further enhanced. BSHXF has been shown to enhance the viability of EPCs, their tube-forming capacity, and the expression of VEGF in vitro. These findings suggest that BSHXF can improve endplate microcirculation and delay IDD progression by maintaining EPCs viability and promoting angiogenesis in endplate.

Financial support

This work was supported by the National Natural Science Foundation of China (grant number 82304955), Shanghai Science and Technology Committee (grant number 20Y11913000), Shanghai Changning District Science and Technology Committee (grant number CNKW2022Y20), Shanghai Changning District Municipal Health Bureau (grant number 2022QN08).

Ethics statement

All animal experiments were conducted in accordance with ethical guidelines and approved by the Laboratory Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine(Ethics approval number: 2021-K-25).

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

The data associated with my study will be made available upon request and deposited into publicly available repositories.

CRediT authorship contribution statement

Yue Xie: Writing – original draft, Methodology, Data curation, Conceptualization. Jianpo Zhang: Writing – original draft, Software, Methodology, Formal analysis. Shengqi Yang: Writing – review & editing, Methodology, Data curation. Weifeng Zhai: Software, Formal analysis. Hailiang Zhao: Methodology, Formal analysis. Zhan Shen: Methodology, Investigation. Ji Guo: Writing – review & editing, Writing – original draft, Supervision, Investigation, Funding acquisition. Yongwei Jia: Supervision, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.