Protective Effects of Changbudodam-tang on Cell Death Signals on the Bone Marrow-Derived Human Mesenchymal Stem Cells via Regulation of MKK7/JNK/c-Jun Signaling Pathway
1Department of Obstetrics and Gynecology, College of Korean Medicine, Daejeon University, Daejeon, Republic of Korea
2Department of Leukopak Research and Developement, QPS Bio-Kinetic, Springfield, MO, USA
*Corresponding Author Ji-Yeon Lee, Department of Obstetrics and Gynecology, College of Korean Medicine, Daejeon University, 62 Daehak-ro, Donggu, Daejeon 34520, Republic of Korea, Tel: +82-42-470-9139, E-mail: jyounl@daum.netAbstract
Objectives
Polycystic ovary syndrome (PCOS) is one of the most common disorders and it shows up to 20% prevalence in reproductive-aged women populations, but no cures are available to date. We aimed to investigate the protective effects of Changbudodam-tang (CBD) on cell death signaling pathways, inflammation, and oxidative stress observed in Bone-Marrow derived human mesenchymal stem cell (BM-hMSC) by means of PCOS therapeutics in the future.
Methods
BM-hMSCs were applied with cell deaths and injuries. Apoptosis and pyroptosis signals were quenched with their related signaling pathways using quantitative PCR, Western blot, and fluorescence image analysis.
Results
Our data clearly displayed hydrogen peroxide- and nigericin-treated cell death signaling pathways via regulations of mitochondrial integrity and interleukin (IL)-1β at the cellular levels (p < 0.01 or 0.001). We further observed that pre-treatment with CBD showed protective effects against oxidative stress by enhancement of antioxidant components at the cellular level, with respect to both protein and mRNA expression levels (p < 0.05, 0.01 or 0.001). The mechanisms of CBD were examined by Western blot analysis, and it showed anti-cell death, anti-inflammatory, and antioxidant effects via normalizations of the Jun N-terminal kinase/mitogen-activated protein kinase kinase 7/c-Jun signaling pathways.
Conclusion
This study confirmed the pharmacological properties of CBD by regulation of cellular oxidation and the inflammation-provoked cell death condition of BM-hMSCs, which is mediated by the MKK7/JNK/c-Jun signaling pathway.
INTRODUCTION
Polycystic ovary syndrome (PCOS) is one of the most common disorders among women of reproductive age, affecting approximately 20% of this population [1]. PCOS leads to various symptoms including hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphological alterations [2, 3]. The pathophysiological mechanism of PCOS remains to be elucidated. Previous studies have reported that complex endocrine dysfunctions and metabolic syndrome mainly contribute to PCOS [4]. These conditions aggravate low to mild chronic inflammation and oxidative stress, contributing to the progression and development of PCOS. Here, the release of pro-inflammatory cytokines leads to an abnormal increase in androgen generation in theca cells [5, 6]. Furthermore, the excessive generation of reactive oxygen species (ROS) exerts oxidative stress by activating ROS-sensitive pathways, such as the c-Jun N-terminal kinases (JNK) signaling pathways [7]. Thus, a combination of anti-inflammatory and antioxidant treatments may be a potent therapeutic strategy for treating patients with PCOS.
Recently, physicians have been recommending lifestyle changes to patients for managing PCOS and metabolic syndrome-related disorders. These changes include a healthy diet, weight loss, and increased physical activity. Another way to manage these disorders may be to take health supplements with antioxidant effects (Ref). In addition, previous studies have reported the utilization of bone marrow human mesenchymal stem cells (BM-hMSC) for enhanced anti-inflammatory effects with immunosuppression in animal models [8, 9].
According to Traditional Oriental Medicine (TOM), herbal medicines and acupuncture treatments are also effective in managing PCOS [10, 11]. Specifically, Changbudodam-tang (CBD) has been traditionally and widely used in the clinic to ameliorate patients’ PCOS symptoms [12ref13-14]. However, the potential anti-inflammatory actions of CBD remain elusive.
In this study, we aimed to investigate the pharmacological effects of CBD on BM-hMSCs under various cell-damaging stimuli, such as cell death signals, inflammatory conditions, and oxidative stress. The potential molecular signaling pathways associated with CBD’s anti-inflammatory and anti-cell injury effects were investigated.
MATERIALS AND METHODS
1. Preparation of Changbudodam-Tang (CBD)
CBD, composed of a total of six types of herbal medicines, was prepared by Daeyeon Pharmaceutical (Incheon, South Korea). The preparation was done in a clean room, following the good manufacturing practices (GMP) approved by the Ministry of Food and Drug Safety (MFDS). The ingredients of CBD and their ratios are listed in Table 1. A total of 54 g of dry CBD ingredients were washed twice using distilled water (DW). The ingredients were then incubated in a dry oven overnight at 60℃ for complete drying needed for extraction. Next, the ingredients were boiled for 3 hours, concentrated for 1.5 hours, and frozen at –80℃ for lyophilization. The resulting CBD powder was then dissolved in DW and centrifuged. The supernatant (20 mg/mL) was filtered using a 0.45 µm syringe applied filter (Millipore) and stored at –80℃ until usage.
2. Cell culture
BM-hMSCs were kindly provided by Dr. Chang-Hyun Gil in the Department of Surgery at Indiana University School of Medicine. The cells were cultured in Xeno-Free BM-hMSC Expansion Media (Sartorius, Goettingen, Germany) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, MO, USA). The cells were seeded in culture dishes of either 60 mm or 35 mm diameter depending on the experiment.
3. Induction of cell death in BM-hMSCs
To investigate the protective effects of CBD against cell death signals, BM-hMSCs were first treated with 200 or 400 µg/mL CBD or 2 ng/mL human transformation growth factor (TGF)-β1 for 6 hours. Next, the cells were treated with hydrogen peroxide, nigericin, or TNF-α. Upon completion of each treatment, the medium was removed and the cells were washed twice with 10 mM phosphate-buffered saline (PBS). Next, the cells were harvested using 0.5% Tris-ethylenediaminetetraacetic acid (EDTA; Invitrogen) and subjected to appropriate experimental procedures for testing.
4. LIVE/DEAD cell assay
BM-hMSCs were seeded in a 35-mm diameter glass bottom dish at 1 × 105 cells/dish. After reaching 60-70% confluence, the cells were treated with the media containing CBD or TGF-β1 for 6 hrs. Next, the cells were treated with 500 µM hydrogen peroxide for 24 hours. The cells were then rinsed twice with PBS and treated with the LIVE/DEAD cell reagents (InvitrogenTM, Cat#. L3224) according to the manufacturer’s protocol. The cells were imaged at 200× magnification using an LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Baden-Wurttemberg, Germany); green fluorescence indicated live cells and red fluorescence indicated dead cells. The captured images were analyzed using Image J version 1.64 (National Institute of Health, MD, USA).
5. Flow cytometry
Flow cytometry (fluorescence assisted cell sorting or FACS, BD Biosciences, NJ, USA) was used to analyze the levels of apoptosis (Annexin V and PI), pyroptosis (PI), cleaved caspase-1 (p10), and interleukin (IL)-1β in the treated cells. For the apoptosis assay, the cells were initially treated with CBD or TGF-β1 for 6 hours, followed by 500 µM hydrogen peroxide treatment for 12 hours. The cells were harvested and stained with PI/Annexin V following the manufacturer’s protocol (Cat#. 88-8005-74, Thermo Fisher). For the pyroptosis assay, the cells were identically treated with CBD or TGF-β1, followed by 3 µM nigericin (3 µM) treatment for 12 hours. The cells were analyzed using a pyroptosis detection kit (Cat#. SKU. 9145, Immunohistochemistry Technology, CA, USA) following the manufacturer’s protocol. For the IL-1β assay, the cells were identically treated with CBD or TGF-β1 and nigericin. The cells were then treated with a permeabilization buffer (0.1% Triton X) for 10 minutes at room temperature (RT), followed by incubation with IL-1β antibody (10 µM Human IL-1 beta/IL-1F2 APC-conjugated antibody, Cat#. IC8406A, R&D Systems, MN, USA).
6. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis
First, mRNA was isolated from the treated cells using the TRIZOL Reagent (Sigma-Aldrich). cDNA was synthesized with 1 µg of the isolated mRNA using a commercial kit. Real-time RT-PCR was performed using the synthesized cDNA and SYBR Green qPCR kit (Cat#. 4368814, Applied BiosystemsTM). The PCR reactions were conducted using Mastercycler RealPlex 2 (Eppendorf, Hauppauge, NY, USA) with a total of 10 µL of final reaction volume (5 μL of SYBR Green Supermix, 2 μL RNase free water, 1 μL of 10 pM primer pairs, and 2 μL of cDNA) (Table 2). The PCR reaction conditions were as follows: 2 minutes at 50℃ for uracil-DNA glycosylase (UDG) activation, 2 minutes at 95℃ for DNA polymerase, and 40 cycles of amplification steps (15 seconds at 95℃, 15 seconds at 60℃, and 15 seconds at 72℃). The mRNA expression levels were analyzed using β-actin as the housekeeping gene and calculated by 2–ΔΔCT.
7. Protein sample preparation
The treated cells were harvested and treated with radio-immunoprecipitation assay (RIPA) buffer containing cocktail proteinase inhibitors and phosphorylated inhibitors (Cat#. ab201119, Abcam). The lysate was centrifuged at 12,000 × g at 4℃ for 60 minutes. The protein concentration in the supernatant was measured via BCA assay (Compat-AbleTM BCA Protein Assay Kit, Cat#. 23229, Thermo Fisher).
8. Protein levels of pro- and anti-inflammatory cytokines in the liver
Enzyme-linked immunosorbent assay kits were used to determine the protein levels of pro- and anti-inflammatory cytokines (BD Biosciences for TNF-α and IL-6 ELSIA; R&D Systems for IL-1β and IL-10 ELISA).
9. Mitochondria integrity
Mitochondrial integrity was analyzed using MitoTracker (Cat#. M7512, MitoTrackerTM Red CMXRos, InvitorgenTM). The deep red fluorescence signals were analyzed using a confocal microscope at 200× magnification.
10. Biochemical assays
The cells were seeded in either a glass-bottom dish of 35 mm diameter or a 96-well microplate at 1 × 105 cells/well and 2 × 103 cells/well, respectively. After treating the cells with either the drug or hydrogen peroxide, 10 µL of 1 µM dichlorodihydrofluorescein diacetate (DCFDA; Cat#. D399, InvitrogenTM) was added and incubated for 30 minutes at 37℃ in the dark.
The presence of ROS was imaged by green fluorescence under a confocal microscope at 200× magnification. For the quantification of ROS level, the cells were lysed with 2N HCl and subjected to UV-Vis spectroscopy (excitation at 488 nm and emission at 535 nm). The lipid peroxidation level was also determined based on the malondialdehyde (MDA) concentration using a commercial kit (Cat#. K739, Bio Vision).
The total thiol level was measured by staining with a fluorescent probe (Cat#. T10096, ThiolTrackerTM, InvitrogenTM) and imaging with a confocal microscope. The total glutathione (GSH) content, SOD activity, and catalase activity in the cell lysates were measured using commercial kits (GSH kit: Cat#. EIAGSHC, Invitrogen; SOD activity kit: Cat#. 19160, Millipore Sigma; Catalase activity kit: EIACATC, Thermo Fisher). A plate reader (Versa Max, SoftMax 5.14) was used for these analyses.
11. Western blot
The protein samples were separated on an SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, Massachusetts, USA). The membrane was incubated in 5% skim milk (in 0.05% PBST buffer) for 1 hour to block non-specific protein binding. The membrane was then incubated with the following primary antibodies: anti-rabbit-MKK7 antibody (CST, #4172), anti-rabbit-phosphorylated MKK7 (Ser271/Thr275) antibody (CST, #4171), anti-rabbit-SAPK/JNK antibody (CST, #9252), anti-rabbit phosphorylated SAPK/JNK (Thr183/Tyr185) (81E11) antibody (CST, #4668), anti-rabbit-c-Jun antibody (CST, #9165), and anti-rabbit-phosphorylated-c-Jun (Ser73)(D47G9)XP® antibody (CST, #3270). Anti-mouse-α-actinin (SCBT, sc-17829, 1:3000) was used as the inner control. After incubating overnight at 4℃, the blots were rinsed thrice with PBST buffer. The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and washed with PBST buffer. The blots were visualized using a chemiluminescent detection reagent (Thermo Fisher Scientific).
12. Statistical analysis
All values were expressed as mean ± standard error of the mean (SEM). The statistical significances of the differences among the groups were analyzed by one-way ANOVA followed by post-hoc multiple comparisons with Tukey t-test using Prism version 9.3.1 (GraphPad 10.01, California, USA). A p-value of less than 0.05 was considered statistically significant.
RESULTS
1. Anti-apoptotic effects of CBD
We first confirmed CBD’s impact on cell viability via LIVE/DEAD cell analysis. As shown in Fig. 1A and B, H2O2 treatment significantly increased the signals corresponding to dead cells (red fluorescence) compared to the control group (p < 0.001). Furthermore, H2O2 treatment also deteriorated the cells’ mitochondrial integrity as shown by the significantly diminished signals (red fluorescence) (p < 0.001 in Fig. 1C, D). These cellular damages were significantly protected by the pre-treated CBD, particularly at 400 µg/mL (p < 0.001 for 400 µg/mL CBD in Fig. 1B and p < 0.001 for both 200 and 400 µg/mL CBD in Fig. 1D).
Flow cytometry indicated that CBD showed a potent anti-apoptotic effect under H2O2-induced stress, as shown by the decreased cell populations in the double-positive quadrant areas corresponding to Annexin V- and PI-stained cells compared to the H2O2-only group (p < 0.01 or 0.001 in Fig. 1E, F).
The treatment of human recombinant TGF-β1, which was used as the positive control, also showed protective effects against H2O2-induced stress compared to the H2O2-only group, as demonstrated by LIVE/DEAD cell analysis, mitochondrial integrity, and Annexin V assay (p < 0.01 or 0.001 in Fig. 1).
2. Anti-pyroptotic effects of CBD
We next examined the protective effects of CBD against pyroptosis, which was induced with nigericin. As expected, the hMSC population in the double-positive area of PI and cleaved caspase-1 (p10) was significantly increased in the nigericin-treated group compared to the control group (p < 0.001 in Fig. 2A, B), indicating the induction of pyroptosis. Pre-treatment of the cells with CBD significantly inhibited pyroptosis, as demonstrated by flow cytometry analysis (p < 0.01 for 200 µg/mL CBD and p < 0.001 for 400 µg/mL CBD in Fig. 2A, B).
As a result of the elevated level of pyroptosis, the cell population in the IL-1β/F2-positive area was significantly increased in the nigericin-treated group compared to the control group (p < 0.001 in Fig. 2C, D). Such elevation of pyroptosis could be significantly inhibited by pre-treating the cells with CBD (p < 0.01 for 200 µg/mL CBD and p < 0.001 for 400 µg/mL CBD in Fig. 2C, D). Both the mRNA and protein levels of IL-1β well supported the observed pyroptosis induced by nigericin treatment (p < 0.001 for both the mRNA and protein expression levels in Fig. 2E, F), as well as the protective effects of CBD (p < 0.01 for 200 µg/mL CBD and p < 0.001 for 400 µg/mL CBD in Fig. 2E, F). The mRNA and protein levels of IL-10, an anti-inflammatory cytokine, were significantly decreased in the nigericin-treated group compared to the control group (p < 0.001 in Fig. 2G, H). Pre-treating the cells with CBD inhibited such reduction in IL-10 (p < 0.001 in Fig. 2G, H).
The positive control group (TGF-β1) showed a potent protective effect against nigericin-induced pyroptosis as demonstrated by flow cytometry, as well as the mRNA and protein expression levels (p < 0.001 in Fig. 2).
3. CBD’s protective effects against cellular oxidative stress
To further investigate the protective effects of CBD against apoptosis and pyroptosis, we assessed CBD’s antioxidant effects. Cellular oxidative stress in hMSCs was significantly induced in the cells treated with H2O2, as demonstrated by staining with DCFDA (intracellular H2O2; green fluorescence) and DHE (superoxide radicals; red fluorescence), compared to the control group (p < 0.001 for DCFDA in Fig. 3A, B; p < 0.01 for DHE in Fig. 3C, D). Pre-treatment with CBD significantly reduced such abnormal increase in ROS induced by H2O2 treatment (p < 0.01 for 200 µg/mL CBD and p < 0.001 for 400 µg/mL CBD for both DCFDA and DHE shown in Fig. 3A-D).
The intracellular level of H2O2 was significantly increased in the H2O2-treated group compared to the control group (p < 0.001 in Fig. 3E). Furthermore, the level of lipid peroxidation, which is the final product of oxidative stress, was also significantly increased in the H2O2-treated group compared to the control group, as demonstrated by MDA measurements (p < 0.001 in Fig. 3F). Pre-treatment with CBD significantly reduced such oxidative damage to the cells (p < 0.01 for 200 µg/mL CBD and p < 0.001 for 400 µg/mL CBD for ROS and MDA shown in Fig. 3E, F).
Human recombinant TGF-β1 treatment significantly decreased all oxidative stress mediators and MDA compared to the H2O2-treated group (p < 0.001 for all parameters shown in Fig. 3).
4. Antioxidant effects of CBD
We further assessed the pharmacological effects of CBD, with a focus on its antioxidant components. The fluorescence imaging analysis indicated that the presence of thiols, which are present in GSH, was significantly decreased upon H2O2 treatment compared to the control group (p < 0.01 in Fig. 4A, B). Pre-treatment with CDB significantly prevented such depletion of thiols observed in the H2O2-treated group (p < 0.001 for both 200 and 400 µg/mL CBD shown in Fig. 4A, B).
The protein level of total GSH content was also significantly decreased by H2O2 treatment (p < 0.001 in Fig. 4C). Pre-treatment with CBD, again, significantly reduced the depletion of GSH compared to the H2O2-treated group (p < 0.01 for 200 µg/mL CBD and p < 0.001 for 400 µg/mL CBD shown in Fig. 4C). The mRNA levels of GSH-related enzymes, such as GSS, GPX3, and GRS, significantly decreased in the H2O2-treated group by 0.35-, 0.41-, and 0.50-fold compared to the control group (p < 0.001 for all enzymes shown in Fig. 4D, F). Pre-treatment with CBD significantly normalized such abnormal gene expression levels (p < 0.05 for 200 µg/mL CBD in GSS; p < 0.001 for others in GSS, GRS, and GPX shown in Fig. 4D-F).
Enzymatic antioxidant components, SOD and catalase, also considerably decreased upon H2O2 treatment. The protein level of SOD significantly decreased in the H2O2-treated group compared to the control group (p < 0.001 in Fig. 4G). The gene expression levels of SOD family members, including SOD1 and SOD2, also significantly decreased in the H2O2-treated group by approximately 0.72-, 0.43-, and 0.87-fold compared to the control group (p < 0.001 for all members shown in Fig. 4H, I). However, the SOD3 level was not significantly altered (p > 0.05 in Fig. 4J). Respective reductions in SOD levels were significantly recovered by pre-treatment with CDB; SOD activities at the protein level and SOD2 at the mRNA level (p < 0.001 for SOD activities and mRNA level of SOD2 shown in Fig. 4G, I). However, SOD1 was not significantly altered (p > 0.05 in Fig. 4H).
The protein level of catalase, an antioxidant enzyme, was significantly decreased in the H2O2-treated group compared to the control group (p < 0.001 in Fig. 4K). The enzyme’s gene expression levels also decreased by 0.54-fold in the H2O2-treated group (p < 0.001 in Fig. 4L). These abnormal alterations were significantly normalized by pre-treatment with CBD (p < 0.01 for 200 µg/mL CBD for catalase activities in Fig. 4K; p < 0.001 for all others in Fig. 4K, L).
The positive control group, TGF-β1, showed strong antioxidant effects with statistically significant differences compared to the H2O2-treated group (p < 0.001 in Fig. 4A-G, I, K, L). However, the mRNA levels of SOD1 and SOD3 were not significantly affected by the treatment (Fig. 4H, J).
5. CBD exerts anti-cell death and antioxidant effects through JNK/MKK7/c-Jun signaling pathways
To examine the underlying mechanisms of CBD’s pharmacological effects on cell death and oxidative stress, we carried out a western blot analysis focusing on the JNK/MKK7/c-Jun signaling pathways. As expected, the protein level of p-JNK was drastically increased by H2O2 treatment compared to the control group. The downstream oxidative stress-induced cell injury and death signaling proteins, such as MKK7 and c-Jun, were also considerably enhanced in the H2O2-treated group. Remarkably, pre-treatment with CBD prevented such phenomena (Fig. 5A). Human recombinant TGF-β1 treatment also showed similar preventive effects (Fig. 5A).
DISCUSSION
BM-hMSC-based therapy is promising for managing PCOS, as this cell type exhibits self-renewing, proliferation, differentiation, and immunomodulatory activities under inflammatory conditions. BM-hMSCs are also highly associated with enhanced ovarian functions by regulating the paracrine signaling pathways. Additionally, the pathological features of PCOS are closely linked to inflammation and oxidative stress. These pathological conditions eventually lead to the activation of cell death signaling pathways. It has been demonstrated that BM-hMSC infusion can reduce inflammation in animals with letrozole (LTZ)-induced PCOS [15, 16]. Based on previous studies, we hypothesized that BM-hMSCs combined with CBD may exhibit enhanced anti-inflammatory and antioxidant activities, thereby preventing the cell death signaling pathways with minimal stimuli.
In line with our expectation, we confirmed that CBD could efficiently inhibit cell death signals, particularly the H2O2-induced apoptosis and nigericin-induced pyroptosis signaling pathways (Figs. 1A, B, E, F, 2A, B). Regarding the protective effects of CBD, the mitochondrial integrity was associated with apoptosis (Fig. 1C, D), while pyroptosis was closely linked to IL-1β, with increases in both the gene and protein levels of IL-10 (Fig. 2C to H). H2O2 is a commonly used chemical for inducing cell death signals in MSCs [17]. Mitochondrial integrity is a critical factor in maintaining cell survival and respiration in biological organisms. When this integrity is altered, it directly leads to the activation of cell death signals, such as caspase signaling pathways and cytochrome c release [18, 19]. Pyroptosis, an inflammation-mediated cell death signaling pathway, is crucial for understanding the relationship between various types of cell death singling and inflammation (particularly the inflammasome signaling pathway) [20, 21]. As a crucial inflammatory cytokine, IL-1β directly mediates pyroptosis under specific conditions [20, 22].
Oxidative stress causes chronic inflammation and is one of the main pathological sources of PCOS [23, 24]. The antioxidant effects of CBD were confirmed by assessing the altered ROS levels via DCFDA and DHE staining (Fig. 3A-D). The decreased intracellular levels of ROS and MDA, the final product of oxidative stress, confirmed the antioxidant effects of CBD (Fig. 3F-G). ROS are harmful free radicals that directly damage the cells and DNA membranes [25]. We suspect that BM-hMSCs’ potent antioxidant activities and culture with CBD may be improving redox homeostasis [26]. Antioxidants are equipped with non-enzymatic and enzymatic components for protection against oxidative damage. GSH is a peptide that quenches free radical adducts through the regulation of its related enzymes [27]. Enzymatic antioxidant components, such as SOD and catalase, play critical roles in preventing severe oxidative stress by removing ROS during cellular oxidation [27, 28]. SOD is an enzyme that converts superoxide to hydrogen peroxide, which is catalyzed by catalase activities. These properties were well demonstrated in BM-hMSCs’ redox status at both the protein and gene levels (Fig. 4A-L).
To investigate the pharmacological actions of CBD against cell death signals, inflammation, and oxidative stress, we performed a western blot analysis focusing on the JNK/MKK7/c-Jun signaling pathways. The cell death signaling pathway resulting from oxidative stress and inflammation is mediated by the MKK7/JNK/c-Jun-singling pathway [29]. In MSCs, JNK plays a critical role in cell differentiation and renewal by cooperating with MKK7 [30]. c-Jun is responsible for osteogenic and adipogenic differentiation in MSCs [31]. However, there is no evidence to explain cell death by cellular oxidation and inflammation.
In this study, the protein level of MKK7 was enhanced by hydrogen peroxide treatment. The phosphorylated JNK and the MKK7/JNK signaling pathway mediate c-Jun activation through phosphorylation, which is a major cell death factor [32]. Pre-treatment with CBD significantly prevented this effect compared to the H2O2-treated group (Fig. 5A).
Despite the high morbidity of PCOS among reproductive women, there is no off-the-shelf drug available. However, the application of MSCs has shown promising potential as an effective treatment option for PCOS. In this study, we examined the therapeutic effects of CBD which is a modified prescription of TOM. The uses of herbal materials are accompanied by several critical issues, such as contamination, original sources, and chemical characterization (Advancing herbal medicine: enhancing product quality and safety through robust quality control practices). To minimize these issues, we used a GMP facility supplied with clearly defined raw materials.
For facile production and material preparation, further studies are needed to elucidate the effects and actions of CBD using pre-clinical animal models. This will involve administering CBD directly and using MSCs pre-cultured with CBD. Furthermore, the MKK/JNK/c-Jun signaling pathways will need to be explored.
CONCLUSION
We explored the potential of BM-hMSC for treating PCOS through anti-inflammatory and antioxidant effects. These effects were associated with various anti-cell death effects. CBD showed potent inhibitory effects against apoptosis and pyroptosis, which are mainly mediated by inflammation and oxidative stress at the cellular level. The underlying mechanisms may be associated with both the inflammation- and oxidation-related signaling pathways, particularly the MKK7/JNK/c-Jun signaling pathway (Fig. 5B).
| Herbal name | Specific name origin | Amount (g) | |||
|---|---|---|---|---|---|
| Atractylodis Rhizom | Atractylodes lancea De Candlle (Vietnam) | 12 | |||
| Cyperi Rhizoma | Cyperus rotundus Linné (Korea) | 12 | |||
| Aurantii Fructus Immatur | Citrus aurantium Linné (China) | 12 | |||
| Citri Unshius Pericarpiu | Citrus reticulata Blanc (Korea) | 6 | |||
| Astragali Radix | Astragalus membranaceus Bunge (China) | 6 | |||
| Cinnamomi Cortex | Cinnamomum cassia Presl (Vietnam) | 6 | |||
| Total | 54 | ||||
| Gene | Species | Primer sequence (forward and reverse) | Application |
|---|---|---|---|
| TNFA | Human | 5′- CTC TTC TGC CTG CTG CAC TTT G-3′ 5′- ATG GGC TAC AGG CTT GTC ACT C-3′ | qPCR |
| IL1B | Human | 5’- CCA CAG ACC TTC CAG GAG AAT G-3’ 5’- GTG CAG TTC AGT GAT CGT ACA GG-3’ | qPCR |
| IL10 | Human | 5’- TCT CCG AGA TGC CTT CAG CAG A-3’ 5’- TCA GAC AAG GCT TGG CAA CCC A-3’ | qPCR |
| GSS | Human | 5′-TAT GTG AGC CGC CTG AAT GCC A-3′ 5′-TGT GAC CTC TCC AGC AGT AGA C-3′ | qPCR |
| GPX3 | Human | 5′-TAC GGA GCC CTC ACC ATT GAT G-3′ 5′-CAC TGA CCT CTA TTG TGG GCT TG-3′ | qPCR |
| GRS | Human | 5′-GTT TAC CGC TCC ACA CAT CCT G-3′ 5′-GCT GAA AGA AGC CAT CAC TGG TG-3′ | qPCR |
| CAT | Human | 5′- GTG CGG AGA TTC AAC ACT GCC A-3′ 5′- CGG CAA TGT TCT CAC ACA GAC G-3′ | qPCR |
| SOD1 | Human | 5′-CTC ACT CTC AGG AGA CCA TTG C-3′ 5′-CCA CAA GCC AAA CGA CTT CCA G-3′ | qPCR |
| SOD2 | Human | 5′-CTG GAC AAA CCT CAG CCC TAA C-3′ 5′-AAC CTG AGC CTT GGA CAC CAA C-3 | qPCR |
| SOD3 | Human | 5′-CAC CAT TGG CAA TGA GCG GTT C-3′ 5′-TGG CTG ATG GTT GTA CCC TGC A-3′ | qPCR |
| β -ACTIN | Human | 5′-GGC ACC ACA CCT TCT ACA ATG A-3′ 5′-AGG TCT TTG CGG ATG TCC ACG T-3′ | qPCR |