Identification and Characterization of Murine Adipose Tissue-Derived Somatic Stem Cells of Shenque (CV8) Acupoint
Acupoints are essential sites on the body’s surface in Traditional Chinese Medicine (TCM), where qi and blood aggregate to support body functions. These points are connected to tissues and organs through meridians, reflecting their physiological or pathological conditions. Acupoints are also used for acupuncture, massage, and other external treatments. Despite their therapeutic use for thousands of years, the mechanisms underlying their effects remain largely unknown. Shenque (CV8), an acupoint located at the navel, has been used in TCM for over 2000 years. However, research into the molecular mechanisms of CV8 acupoint therapy has lagged behind its clinical application. This study aimed to explore the mechanisms of umbilical acupoint therapy by investigating the role of somatic stem cells derived from the CV8 acupoint.
Gross Anatomical and Histomorphological Observations of CV8 Acupoint
The CV8 acupoint is anatomically located at the navel in the linea alba of mice. Gross observations revealed a patch-like distribution of adipose tissues at the CV8 acupoint. Under a stereomicroscope, these adipose tissues formed a ball-like fat mass. Histomorphological analysis using hematoxylin and eosin (H&E) staining showed that the center of the navel in mice was composed of Oil Red O-positive tissues rich in adipose cells. Masson staining further confirmed the presence of connective tissue surrounding the adipose cells. These findings suggest that the CV8 acupoint is rich in adipose tissue, which may play a role in its therapeutic effects.
Immunohistochemical Staining of CV8 Acupoint Tissues
Immunohistochemical staining was performed to identify the presence of stem cell antigen-1 (Sca-1) positive cells in the CV8 acupoint tissues. Sca-1, a marker for somatic stem cells, was found to be distributed in various sections of the CV8 acupoint tissues. This indicates that the CV8 acupoint contains a population of somatic stem cells, which may contribute to its therapeutic properties.
Isolation and Culture of Somatic Stem Cells from CV8 Acupoint
Adipose tissues from the CV8 acupoint and non-acupoint sites in the groin were isolated and cultured. Cells from both sites adhered to the culture plates after 3 days and reached 80% confluence after 7–10 days. The cells exhibited a long fusiform and fibroblast-like morphology after passaging to the third generation (P3). Flow cytometry analysis revealed high expression of stem cell markers Sca-1 and CD44, and low expression of CD31 and CD34 in both groups. These results confirm that the cells isolated from the CV8 acupoint and non-acupoint sites are somatic stem cells.
Proliferation Ability of Somatic Stem Cells
The proliferation ability of somatic stem cells from the CV8 acupoint and non-acupoint sites was compared using the cell counting kit-8 (CCK-8) method. Both groups exhibited an S-shaped proliferation curve, with a slow growth phase in the first 72 hours, followed by a logarithmic growth phase from day 3 to day 5, and a plateau phase from day 5 to day 7. However, the proliferation rate of somatic stem cells from the CV8 acupoint was significantly higher than that of non-acupoint cells from day 4 onwards. Specifically, on day 4, the absorbance values were 0.5138 ± 0.0111 for CV8 acupoint cells versus 0.4107 ± 0.0180 for non-acupoint cells (t = 8.447, P = 0.0011). This trend continued on day 5 (0.6890 ± 0.0070 vs. 0.5520 ± 0.0118, t = 17.310, P < 0.0001), day 6 (0.7320 ± 0.0090 vs. 0.6157 ± 0.0123, t = 13.190, P = 0.0002), and day 7 (0.7550 ± 0.0050 vs. 0.6313 ± 0.0051, t = 42.560, P < 0.0001). These findings suggest that somatic stem cells from the CV8 acupoint have enhanced proliferative capabilities compared to those from non-acupoint sites.
Differentiation Potential of Somatic Stem Cells
The differentiation potential of somatic stem cells from the CV8 acupoint and non-acupoint sites was evaluated in terms of adipogenic, osteogenic, and neurogenic stem cell-like cell differentiation.
Adipogenic Differentiation
Adipogenic induction was performed, and the presence of lipid droplets was confirmed by Oil Red O staining. The percentage of Oil Red O staining area was significantly higher in CV8 acupoint cells (9.224 ± 0.345%) compared to non-acupoint cells (3.933 ± 1.800%) (t = 5.000, P = 0.0075). This indicates that somatic stem cells from the CV8 acupoint have a greater capacity for adipogenic differentiation.
Osteogenic Differentiation
Osteogenic differentiation was assessed using Alizarin Red S staining to detect mineralized nodules. The percentage of mineralization area was significantly lower in CV8 acupoint cells (2.697 ± 0.627%) compared to non-acupoint cells (7.254 ± 0.958%) (t = 6.893, P = 0.0023). This suggests that somatic stem cells from the CV8 acupoint have a reduced capacity for osteogenic differentiation.
Neurogenic Stem Cell-Like Cell Differentiation
Neurogenic differentiation was evaluated by observing the formation of neural stem cell-like cell clusters, or neurospheres. The number of neurospheres was significantly higher in CV8 acupoint cells compared to non-acupoint cells across different diameter ranges: <50 µm (7.2000 ± 1.3040 vs. 2.6000 ± 0.5477, t = 7.273, P 100 µm (2.6000 ± 0.5477 vs. 0.8000 ± 0.8367, t = 4.025, P = 0.0038). This indicates that somatic stem cells from the CV8 acupoint have a greater capacity for neurogenic stem cell-like cell differentiation.
Discussion
The CV8 acupoint, located at the navel, has been used in TCM for over 2000 years to treat various conditions. However, the mechanisms underlying its therapeutic effects remain poorly understood. This study provides novel insights into the role of somatic stem cells in the therapeutic effects of the CV8 acupoint.
The findings reveal that the CV8 acupoint is rich in adipose tissue, which contains a population of somatic stem cells. These cells exhibit enhanced proliferative capabilities and a greater capacity for adipogenic and neurogenic stem cell-like cell differentiation compared to somatic stem cells from non-acupoint sites. However, they have a reduced capacity for osteogenic differentiation. These properties suggest that somatic stem cells from the CV8 acupoint may play a role in tissue repair and regeneration, particularly in adipose and neural tissues.
The enhanced adipogenic differentiation of CV8 acupoint cells may be related to the high adiponectin content observed in serum after moxibustion at the CV8 acupoint. Adiponectin is known to regulate metabolic processes and may contribute to the therapeutic effects of navel therapy. Similarly, the enhanced neurogenic differentiation potential of CV8 acupoint cells may be relevant to the treatment of neurological conditions, as somatic stem cells have been shown to migrate to lesion sites and differentiate into neural cells.
The reduced osteogenic differentiation capacity of CV8 acupoint cells suggests that these cells may not be as effective in bone repair. However, this does not diminish their potential therapeutic value, as their enhanced adipogenic and neurogenic differentiation capabilities may be more relevant to the conditions treated by navel therapy.
Conclusion
This study demonstrates that the CV8 acupoint is rich in adipose tissue containing somatic stem cells with unique biological properties. These cells exhibit enhanced proliferation, adipogenic differentiation, and neurogenic stem cell-like cell differentiation, but reduced osteogenic differentiation. These findings provide new insights into the mechanisms of umbilical acupoint therapy and suggest that somatic stem cells from the CV8 acupoint may play a key role in its therapeutic effects. Future research should focus on in vivo experiments to further validate these findings and explore the potential applications of CV8 acupoint somatic stem cells in regenerative medicine.
doi.org/10.1097/CM9.0000000000001850
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