The role of mesenchymal stem cells in allergic rhinitis and its relationship with IL-10, plasma cells and regulatory T cells.

Aim Allergic rhinitis (AR) is an IgE-mediated inflammation of the nose. Regulatory T cells (Tregs), plasma cells and inflammatory cytokines have shown to play a critical role in allergic airway inflammation. The aim of the study was to investigate the role of mesenchymal stem cells (MSCs) in generating Treg cells and plasma cells associated with regulating interlukin-10 (IL-10) in AR model. Methods Fifteen male Wistar rats (6 to 8 weeks old) were randomly divided into three groups (control group, sham group, and MSCs treatment group). Ovalbumin (OVA) nasal challenge was conducted daily from day 15 to 21, and MSCs (1x106) were administrated intraperitoneally to OVA-sensitized rats on day 21. Sneezing was observed from day 24 to 27. The rats were sacrificed on day 24 and day 27. The expression of Treg and plasma cells was analysed by flow cytometry assay. The level of IL-10 was analysed under ELISA assay. Results This study showed that the percentage of sneezing and rubbing times significantly decreased in MSCs treatment associated with the regulation of IL-10 level and plasma cell. This finding was aligned with the significant increase of Treg level. Conclusion MSCs administration regulates IL-10 and plasma cell-mediated immune and inflammatory responses while increasing Treg cell production. MSCs may be a promising therapeutic target for treating Treg-mediated allergic diseases.

Secretome of hypoxia-preconditioned mesenchymal stem cells enhance the expression of HIF-1a and bFGF in a rotator cuff tear model.

Aim To determine the effect of secretome hypoxia mesenchymal stem cells (SH-MSCs) on the relative gene expression of hypoxia inducible factor-1a (HIF-1a) and basic fibroblast growth factor (bFGF) in accelerating histomorphometric repair of tendon to bone interface healing in rats acute rotator cuff tear (RCT) model. Methods This is experimental research with posttest control group design. Thirty-male Wistar rats were divided into five treatment groups: healthy group and rotator cuff reconstruction group included four groups: SH-MSCs W2 (the treatment group was given a SH-MSCs 0.5 mL and terminated at weeks 2), NaCl W2 (the control vehicle group was given NaCl 0.5 mL and terminated at weeks 2), SH-MSCs W8 (the treatment group was given a SH-MSCs 0.5 mL and terminated at weeks 8), and NaCl W8 (the control vehicle group was given NaCl 0.5 mL and terminated at weeks 8). On the termination day, all the rats were terminated and HIF-1a and bFGF gene expression were analysed using qRT-PCR. Results SH-MSCs significantly increased the HIF-1a and bFGF gene expression than NaCl group even in week 2 and week 8. The highest increased gene expression of HIF-1a and bFGF was on week 8. Conclusion SH-MSCs are important in the healing repair process of tendon-to-bone interface in acute RCT model rats through increasing gene expression of HIF-1α and bFGF.

Red algae extract suppresses caspase-3 gene expression and induces catalase antioxidant enzyme in testicles of rats induced by boric acid.

Aim To determine the effect of red algae extract on the gene expression of catalase and caspase-3 in testicules of rats induced by boric acid (BA). Methods This is experimental research with post-test control group design. Twenty four healthy male Wistar rats were divided into four treatment groups: a healthy group, negative control group, two treatment groups with red algae extract 400mg/kgBW/day (T1) and red algae extract 800mg/kgBW/day (T2). Each group was treated with BA 500mg/kgBW/day for 14 days, whereas the healthy group did not receive BA. In the treatment groups T1 and T2 were given red algae extract for 14 days. On day 15 all treatment groups were terminated and catalase and caspase-3 gene expression were analysed using qRT-PCR. Results In the healthy group, the expression of the catalase gene was 1.39±0.67 and the expression of the caspase-3 gene was 1.06±0.17. In the negative control group, there was a significant decrease in catalase gene expression, 0.68±0.27 (p<0.05), and a significant increase in caspase-3 gene expression, 5.71±2.47 (p<0.05). Treatment groups T1 and T2 showed a significant increase in catalase gene expression, 2.67±0.69; and 2.85±0.64, respectively (p<0.05) and caspase-3, 3.96±1,16 and 1.89±0.84, respectively, compared to the control group. Conclusion: The administration of red algae extract had a significant effect on increasing the expression of the catalase gene and decreasing the expression of the caspase-3 gene. This suggests that red algae extract has the potential to be developed as a protective agent against exposure to the effects of BA.

Anti-inflammatory effect of Alpinia galanga extract on acute inflammatory cell model of peripheral blood mononuclear cells stimulated with TNF-α.

Aim Studies have shown that SARS-Cov-2 has the ability to activate proinflammatory cytokine leading to acute inflammation. During the SARS-Cov-2 infection, an increase of the secretion of production TNF-α in seen in COVID-19 patients along with a decrease in anti-inflammatory cytokine IL-10, and growth factor TGF-ß caused cytokine storm and damaged tissues. Alpinia galanga extract contains several secondary metabolites with strong antiinflammation and antioxidant effect. The aim of this study was to evaluate the effect of Alpinia galanga extract on peripheral blood mononuclear cells (PMBC) acute inflammation cells model stimulated with TNF-α. Methods Alpinia galanga was extracted under maceration methods on ethanol 96%. The PMBC was collected from three healthy humans and isolated using ficol reagent and cultured with the TNF-α 100pg/mL for 72 h. The TNF-α levels were evaluated under ELISA reader. Furthermore, the IL-10 and TGF-ß gene expression was analysed using qRT-PCR after 24 h treatment with Alpinia galanga extract. Results Alpinia galanga extract has no cytotoxic effect on Vero cells with IC50 value of >1000µg/mL. The PBMC acute inflammation cells model stimulated by TNF-α 100pg/mL, after 72 h induction the PBMC cells significantly expressed a high level of TNF-α up to 341±10.87 pg/mL. Furthermore, the treatment of Alpinia galanga significantly increased the anti-inflammatory cytokine IL-10 and growth factor TGF-ß in dose dependent manner. Conclusion These findings suggested that Alpinia galanga extract has strong antiinflammation activity.

Hypoxic secretome mesenchymal stem cells inhibiting interleukin-6 expression prevent oxidative stress in type 1 diabetes mellitus.

Aim Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the chronic inflammation of the pancreatic islets of Langerhans. Hyperglycaemia leads to suppressed antioxidant enzyme and increased inflammation in the pancreatic cell, resulting in pancreatic cell death. Hypoxic secretome mesenchymal stem cells (HS-MSCs) are soluble molecules secreted by MSCS that have the antiinflammation ability by secreting various cytokines including IL-10 and TGF-ß which potent as a promising therapeutic modality for T1DM. This study aims to investigate the role of HS-MSCs in regulating superoxide dismutase (SOD) and caspase-3 gene expression in T1DM model. Methods Twenty male Wistar rats (6 to 8 weeks old) were randomly divided into four groups (sham, control, HS-MSCs 0.5 mL and HS-MSCs 1 mL intraperitoneal treatment group). Streptozotocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs 0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intraperitoneally on day 7, 14, and 21 after STZ administration. The rats were sacrificed on day 28; the gene expression of SOD and IL-6 was analysed by qRT-PCR. Results This study showed that the ratio of SOD significantly increased in HS-MSCs treatment associated with suppression of IL-6 gene expression. Conclusion HS-MSCs administration suppresses oxidative stress and inflammation by up regulating SOD and inhibiting IL-6 to control T1DM.

Combination Curcuma longa and Phyllanthus niruri Extract Potentiate Antiproliferative in Triple Negative Breast Cancer MDAMB-231 Cells.

BACKGROUND: Triple negative breast cancer cells (TNBC) are a small part of cancer-inducing cells in breast cancer, which are characterized by high metastatic and self-renewal. Self-renewal has the ability to renew itself and loses control of proliferation. Curcuma longa extract (CL) and Phyllanthus niruri extract (PN) known to have anti-proliferative effects on cancer cells. However, the effects of combination CL and PN on TNBC proliferation still unclear. AIMS: This study aimed to evaluate the antiproliferative effects of the combination CL and PN on TNBC MDAMB-231 and attempted to elucidate the underlying molecular mechanisms. SUBJECTS AND METHODS: The dried rhizomes of Curcuma longa and the herbs of Phyllanthus niruri were macerated with ethanol for 72 h.The antiproliferative and synergistic effects of combination CL and PN were investigated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Combination index values were calculated using CompuSyn (ComboSyn, Inc, Paramus, NJ). The cell cycle and apoptosis assay were determined by propidium iodide (PI) and PI-AnnexinV assay under flow cytometer, respectively. The intracellular ROS levels were evaluated using 2',7'-Dichlorodihydrofluorescein diacetate (DCFDA) assay. The mRNA expressions of proliferation-related genes in the cells were determined using bioinformatic assay. RESULTS: The CL and PN single treatment caused a potent and dose-dependent decrease in the percentage of viable cells with IC50 value of 13 µg/mL and 45 µg/mL for 24 h, respectively. The combination index values of the different combinations ranged from 0.08 - 0.90, indicating slightly strong to very strong synergistic effects. The combination of CL and PN also remarkably induced the S- and G2/M-phases cell cycle arrest that leading to apoptosis induction. Furthermore, the combination of CL and PN treatment induced the intracellular reactive oxygen species (ROS) levels. Mechanistically, the AKT1, EP300, STAT3 and EGFR signaling as potential targets of combination CL and PN in antiproliferation and antimetastatic of TNBC. CONCLUSIONS: The combination of CL and PN exerted promising antiproliferative effects in TNBC. Therefore, CL and PN may be considered a potential source for the development of potent anticancer drugs for breast cancer treatment.

In vitro synergistic effect of hesperidin and doxorubicin downregulates epithelial-mesenchymal transition in highly metastatic breast cancer cells.

BACKGROUND: We previously reported that in highly metastatic breast cancer cells, doxorubicin (DOX) at non-toxic concentrations promoted cell migration and invasion. Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavonoid glycoside isolated from citrus/lemon plant that possesses a cytotoxic effect in several cancer cells. In this study, we investigate whether DOX efficacy is enhanced by hesperidin (Hsd) and the molecular pathway involved in highly metastatic breast cancer, 4T1. METHODS: Combined cytotoxicity of Hsd and DOX was evaluated with MTT assay and was analyzed using Chou-Talalay's method. To better understand the underlying mechanism, several factors, including apoptosis and cell cycle arrest were analyzed by flow cytometry. In addition, antimigration activity was evaluated by scratch wound healing assay, MMP-9 expression by ELISA and gelatin zymography, and Rac-1 protein level using western blot. The data on survival rate and expression level of MMP-9 and Rac-1 were obtained from Gene Expression OMNIBUS (GEO). RESULTS: Under MTT assay, Hsd showed a cytotoxic effect in a concentration-dependent manner with an IC50 value of 284 µM on 4T1 cells. Hsd synergistically enhanced the cytotoxic effect of DOX which seemed to correlate with an increase in apoptotic cell death, G2/M cell cycle arrest and blocked the migration of 4T1 cells. At 10 nM, doxorubicin induced lamellipodia formation, and increased the level of Rac-1 and metalloproteinase-9 (MMP-9) expression. Interestingly, combined treatment of DOX and Hsd dramatically downregulated the expression of MMP-9 and Rac-1. These results indicated that Hsd block the cell migration induced by DOX under in vitro studies. CONCLUSION: These findings strongly suggest that Hsd possesses a potential synergistic effect that can be developed to enhance the anticancer efficacy of DOX and reduce the risks of chemotherapy use in highly metastatic breast cancer.

Clitorea ternatea flower extract induces platelet-derived growth factor(PDGF) and GPx gene overexpression in ultraviolet (UV) B irradiationinduced collagen loss.

Aim To determine the effect of Clitorea ternatea flower extract (CTFE) in the gel dosage form on the expression of GPx and platelet-derived growth factor (PDGF) in ultraviolet (UV) - B irradiation-induced collagen loss rat model. Methods This is experimental research with post-test control group design. Twenty healthy male Wistar rats were divided into four treatment groups: a sham group, UVB control group, two treatment groups with gel of CTFE 5% and gel of CTFE 10%, respectively. Each group was treated with UVB at 302 nm with a MED of 160 mJ/cm2 for 5 days, whereas the sham group did not receive UVB. In the treatment groups CTFE 5% and CTFE 10% gel were given on the 6th to the 14th day. On day 14 all treatment groups were terminated, and GPx and PDGF gene expression were analysed using qRT-PCR. Results In the group of gel of CTFE 10%, there was a significant increase in GPx gene expression (9.51±1.83) and PDGF (4.36±1.18) compared to the UVB control group which had GPx and PDGF gene expression of 4.90±1.64) and 0.032±0.01, respectively. Conclusion The administration of CTFE gel showed an increase of the expression of GPx and PDGF g.

Regulation of p53 and survivin by Curcuma longa extract to caspase-3 dependent apoptosis in triple negative breast cancer cells.

Aim Triple negative breast cancer cells (TNBC) are the population of breast cancer cells that are responsible for cancer recurrence and apoptosis resistance. Unfortunately, current therapies have limited efficacy to TNBC population due to apoptosis resistance and chemoresistance. Tumour suppressor p53 and survivin are primary targets for TNBC therapy. Consequently, a search for a natural compound which targets p53 and survivin is needed to further advance TNBC treatment. Curcuma longa extract (CL), a natural compound induces apoptosis in several cancer cells by targeting various molecules and possess fewer side effects. However, a possible potential of CL as p53- and survivin modulating agent in TNBC cells has not been investigated. Methods MDAMB-231 cells were treated with several concentration of CL, after which, viability, p53 gene expression, surviving protein expression, and caspase-3 protein expression were evaluated. Results After 24-h treatment, CL possessed cytotoxic effect with IC50 value of 13 µg/mL. Treatment with 1.625, 3.25, 6.5, and 13 µg/mL of CL resulted in 2.70-25.80% increase in caspase-3 expression levels followed by 94.60 - 21.60% decrease in survivin protein levels. CL induced remarkably p53 gene expression ratio up to 5-fold at 13 µg/mL. Survivin protein levels were inversely proportional to p53 accumulation levels. Low survivin protein levels combined with high levels of p53 accumulation were correlated to higher apoptotic rates. Conclusion p53 and survivin as molecular targets of CL contribute to caspase-3-dependent apoptosis in TNBC cells and this compound represents an attractive p53- and survivin modulating agent in TNBC.

Revealing the decrease of indoleamine 2,3-dioxygenase as a major constituent for B cells survival post-mesenchymal stem cells co-cultured with peripheral blood mononuclear cell (PBMC) of systemic lupus erythematosus (SLE) patients.

Aim Mesenchymal stem cells (MSCs) have potent immunosuppressive properties to control systemic lupus erythematosus (SLE) disease by inhibiting indoleamine 2,3-dioxygenase (IDO), and increasing regulatory T cells (Treg) to control innate and adaptive immune cells. However, the interaction and mechanism regarding IDO and B cells in the co-culture of MSC and SLE peripheral blood mononuclear cell (PBMCs) remain unclear. This study aimed to investigate the effects of MSCs in controlling B cells through IDO expression in PBMC of SLE patients. Methods This study used a post-test control group design. MSCs were obtained from human umbilical cord blood and characterized according to their surface antigen expression and multilineage differentiation capacities. PBMCs isolated from SLE patients were divided into five groups: sham, control, and three treatment groups. The treatment groups were treated by co-culturing MSCs to PBMCs with a ratio of 1:10, 1:25, and 1:40 for 72 h incubation. The B cell levels were analysed by flow cytometry with cytometric bead array (CBA) and the IDO levels were determined by ELISA. Results The percentages of B cells decreased significantly in groups treated by dose-dependent MSCs, particularly in T1 and T2 groups. These findings were aligned with the significant decrease of the IDO level. Conclusion MSCs control B cells-mediated by a decrease of IDO in PBMC of SLE patients.

Rats' umbilical-cord mesenchymal stem cells ameliorate mast cells and Hsp70 on ovalbumin-induced allergic rhinitis rats.

Aim Allergic rhinitis (AR) is a heterogeneous condition that has been associated with inflammatory responses and is characterized by clinical typical symptoms of nasal itching, sneezing, watery discharge and congestion. Mesenchymal stem cells (MSCs) are multipotent stem cells that have the immunoregulatory ability by secreting various cytokines which potent as a promising therapeutic modality for allergic airway diseases, including AR. The aim of this study was to investigate the effect of rat UC-MSCs on the number of mast cells, the expression of Hsp70 indicated by the nasal symptoms allergic, particularly nasal rubbing in ovalbumininduced AR rats. Methods Fifteen male Wistar rats (6 to 8 weeks old) were randomly divided into three groups (control group, sham group, and OVA+MSCs group). OVA nasal challenge was conducted daily from day 15 to 21, and UC-MSCs (1x106 ) were administrated intraperitoneally to OVA-sensitized rats on day 21. Nasal rubbing was observed from day 22 to 28. The rats were sacrificed on day 22 and day 28. The nasal cavity tissues were prepared for histological observations. Results The administration of UC-MSCs could reduce the number of mast cells and the expression of Hsp70 leading to reduction of nasal symptoms allergic, particularly nasal rubbing. Conclusion Based on this finding, MSCs present a promising immediate curative effect to the inflammatory reaction in AR rats.

The CD4+CD25+FoxP3+ Regulatory T Cells Regulated by MSCs Suppress Plasma Cells in a Mouse Model of Allergic Rhinitis.

BACKGROUND: Allergic Rhinitis (AR) is the most common immunological disease that has been associated with inflammatory responses and is characterized by sneezing. Previous studies found that AR's allergen exposure significantly induces plasma cells and reduces regulatory T (Treg) cells, a population that contributes to control AR. Therefore, upregulating Treg expression can regulate plasma cells leading to inhibit sneezing in AR. Mesenchymal stem cells (MSCs) are multipotent stem cells that have the immunoregulatory and antiinflammation ability by secreting various cytokines including IL-10 and TGF-ß which potent as a promising therapeutic modality for allergic airway diseases, including AR. OBJECTIVE: To investigate the role of MSCs in generating CD4+, CD25+, and Foxp3+ Regulatory T cells associated with suppressing plasma cell in AR model. METHODS: In this study, fifteen male Wistar rats (6 to 8 weeks old) were randomly divided into three groups (control group, sham group, and MSCs treatment group). OVA nasal challenge was conducted daily from day 15 to 21, and MSCs (1x106) were administrated intraperitoneally to OVA-sensitized rats on day 21. Sneezing was observed from day 22 to 28. The rats were sacrificed on day 22 and day 28. The expression of CD4+ CD25+ Foxp3+ in Treg and plasma cells was analyzed by flow cytometry assay. RESULTS: This study showed that the percentage of plasma cell and sneezing times significantly decreased in MSCs treatment. This finding was aligned with the significant increase of CD4+CD25+Foxp3+ Treg level. CONCLUSION: MSCs administration suppress plasma cells population and sneezing times by up regulating Treg to control AR.

Cell Cycle Modulation of CHO-K1 Cells Under Genistein Treatment Correlates with Cells Senescence, Apoptosis and ROS Level but in a Dose-Dependent Manner.

Purpose: Genistein, a soy isoflavone, exhibits a biphasic effect on cells proliferation with some different effects between ER-alpha and ER-beta. The objective of this present study is to determine the modulatory effect based on cell cycle progression under genistein treatment in combination with 17-ß estradiol (E2) on CHO-K1 cells. Methods: The effect of genistein 0.1-100 µM on cells proliferation was examined by MTT assay. The modulation of genistein and estradiol (E2) on cell cycle and apoptosis were observed by using flowcytometry with PI and PI/AnnexinV staining, respectively. Moreover, the effect of genistein and E2 on senescence cells, and ROS level were determined by senescence-associated ß-galactosidase (SA ß-gal) staining and by using flowcytometry with 2', 7'-dichlorofluorescin diacetate (DCFDA) staining, respectively. The expression level of the cell cycle and senescence protein markers were observed by immunoblotting. Results: Single treatment of genistein at physiologically achievable (low) concentration (<2 µM) induced proliferation of CHO-K1 cells while at a pharmacological (high) concentration (50 and 100 µM) suppressed cells proliferation. Interestingly, treatment of genistein at the physiological concentration in combination with E2 for 24, 48 and 72 h decreased cells viability on CHO-K1 cells compared to untreated cells. Further analysis of the cells showed that 50 µM genistein induced G2/M phase accumulation and induced apoptosis. Moreover, genistein induced cell senescence and increased ROS level. Immunoblotting analysis showed the decreasing of ERalpha, Bcl2, and ppRb protein level upon treatment of 1 µM Gen and 1 nM E2. Conclusion: Our results suggest that the cell proliferation inhibitory mechanism of genistein at pharmacological concentration involved the induction of cell senescence, and the elevation of ROS level. Moreover, the decreased of cells proliferation upon treatment of physiological concentration of genistein in combination with E2 may be correlated with the alteration of ER expression.