Objective To explore the effect of rehmanniae radix extract(RRE)on chondrocyte apoptosis in the rabbit model of knee osteoarthritis(KOA)by regulating the miR-485-5p/heat shock protein 90 beta family member 1(Hsp90b1)axis.Methods New Zealand rabbits were randomly assigned into control,KOA,low-dose RRE,medium-dose RRE,high-dose RRE,celecoxib,high-dose RRE+antagonist control,and high-dose RRE+miR-485-5p antagonist groups,with 12 rabbits in each group.Rabbits in other groups except the control group were modeled for KOA with the improved Hulth method.After modeling for 8 weeks,the rabbits were administrated with corresponding agents for 4 weeks.The changes in the activity rating of rabbits were recorded.ELISA was employed to measure the levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the serum.Safranine O-fast green staining was conducted to reveal the pathological changes in the cartilage tissue and Mankin scoring was performed.TUNEL was employed to detect chondrocyte apoptosis.Real-time fluorescence quantitative PCR was performed to determine the expression of miR-485-5p in the cartilage tissue.Western blot was employed to determine the protein levels of Hsp90b1,cleaved cysteinyl aspartate-specific proteinase-3(Caspase-3),and Bcl2-associated-X(Bax)in the cartilage tissue.The dual-luciferase reporter assay was employed to examine the relationship between miR-485-5p and Hsp90b1.Results Compared with the control group,the KOA group showed down-regulated expression of miR-485-5p,elevated levels of TNF-α and IL-6 in the serum,cartilage erosion and losses,and increases in activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.001).Compared with the KOA group,RRE at low,medium,and high doses,and celecoxib up-regulated the expression of miR-485-5p,lowered the levels of TNF-α and IL-6 in the serum,alleviated the pathological damage to the cartilage tissue,and decreased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).Compared with the high-dose RRE group and the high-dose RRE+antagonist control group,high-dose RRE+miR-485-5p antagonist down-regulated the expression of miR-485-5p,elevated the levels of TNF-α and IL-6 in the serum,exacerbated the pathological damage to the cartilage tissue,and increased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).The results indicated that there was a targeted regulatory relationship between miR-485-5p and Hsp90b1.Conclusion RRE may inhibit the expression of Hsp90b1 by up-regulating miR-485-5p,thereby inhibiting chondrocyte apoptosis in the rabbit model of KOA.
Animals ; Rabbits ; Apoptosis/drug effects* ; Chondrocytes/pathology* ; Osteoarthritis, Knee/drug therapy* ; MicroRNAs/metabolism* ; Rehmannia/chemistry* ; Disease Models, Animal ; Tumor Necrosis Factor-alpha/blood* ; Plant Extracts/pharmacology* ; Interleukin-6/blood* ; HSP90 Heat-Shock Proteins/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology*

Objective To investigate the role and mechanism of afzelin(AFZ)in treating Crohn's disease-like colitis.Methods A mouse model of 2,4,6-trinitrobenzene sulfonic acid-induced colitis was established to assess the effect of AFZ on experimental colitis in vivo.A Caco-2 cell model of tumor necrosis factor(TNF)-α-induced inflammation was established to evaluate the effects of AFZ on the intestinal barrier function,intestinal epithelial cell apoptosis,and mitochondrial function in vitro.The animal and cell experiments were performed to validate the regulatory role of the adenosine monophosphate-activated protein kinase(AMPK)/silent information regulater 1(SIRT1)/peroxisome proliferator-activated receptor gamma coactivator(PGC)-1α pathway in the treatment of colitis with AFZ.Results AFZ reduced the disease activity index(P=0.003),weight loss(P<0.001),colon shortening(P<0.001),inflammation score(P=0.002),pro-inflammatory cytokine release(interleukin-6:P<0.001;TNF-α:P=0.010),and intestinal barrier permeability(fluorescein isothiocyanate dextran 4:P<0.001;intestinal-type fatty acid-binding protein:P=0.013).Meanwhile,AFZ increased the colonic transepithelial electric resistance(P=0.001),reduced bacterial translocation(P<0.001),and promoted the localization and up-regulated the expression of tight junction proteins [zonula occluden-1(P=0.005) and Claudin-1(P=0.024)].AFZ exerted a protective effect on the Caco-2 cells exposed to TNF-α in terms of intestinal epithelial cell permeability(P=0.017),transepithelial electric resistance(P=0.014),and tight junction protein[zonula occluden-1(P=0.014) and Claudin-1(P=0.006)] localization and expression.Furthermore,the cell and animal experiments confirmed that AFZ reduced the percentage of apoptosis(P<0.001,P=0.013)and the expression of cleaved-caspase 3(P=0.028,P=0.004)and Bax(P=0.004,P=0.020),and upregulated the Bcl2(P=0.020,P=0.006)level in intestinal epithelial cells.Additionally,AFZ increased the number of mitochondria,mitochondrial membrane potential,and copy number of mitochondrial DNA(P=0.007)in intestinal epithelial cells,while enhancing the activities of mitochondrial respiratory chain complex Ⅰ(P=0.005)and complex Ⅳ(P=0.001).The activation of the AMPK/SIRT1/PGC-1α pathway was involved in the protective effects of AFZ on mitochondrial function and apoptosis in intestinal epithelial cells.Conclusion AFZ alleviates mitochondrial dysfunction and apoptosis in intestinal epithelial cells by activating the AMPK/SIRT1/PGC-1α pathway,thereby ameliorating intestinal barrier dysfunction and experimental colitis.
Animals ; Colitis/drug therapy* ; Humans ; Caco-2 Cells ; Mice ; Trinitrobenzenesulfonic Acid ; Apoptosis/drug effects* ; Disease Models, Animal ; AMP-Activated Protein Kinases/metabolism* ; Sirtuin 1/metabolism*

OBJECTIVE: To analyze the influences of dihydromyricetin on the proliferation and apoptosis of chondrocytes in osteoarthritis induced by hydrogen peroxide (H₂O₂) through reactive oxygen species (ROS)/p38 mitogen activated protein kinase (p38-MAPK) pathway. METHODS: Five C57BL/6J mice were euthanized by cervical dislocation after anesthesia. Chondrocytes were extracted and cultured.After passage, the chondrocytes were divided into control group, H2O2 group (0.8 μmol·L^-1 H₂O₂), dihydromyricetin low concentration group (0.8 μmol·L^-1 H₂O₂+20 μmol·L^-1 dihydromyricetin), dihydromyricetin high concentration group (0.8 μmol·L^-1 H₂O₂+80 μmol·L^-1 dihydromyricetin), and ROS inhibitor N-acetylcysteine (NAC) group (0.8 μmol·L^-1 H₂O₂+5 mmol·L^-1 NAC). The activity of chondrocytes was measured by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of chondrocytes was measured by Hoechst 33342 method. The level of ROS in chondrocytes was measured by 2, 7-dichlorofluorescein diacetate (DCFH-DA) fluorescence probe.The level of Type II collagen α1 (Col2α1) mRNA was measured by qRT-PCR.And the expression of Col2α1, p-p38-MAPK/p38-MAPK, B cell lymphoma gene-2 (Bcl-2) and Bcl-2 associated X protein (Bax) proteins was detected by Western blot. RESULTS: The chondrocytes showed swirling fibrous mass, and the expression of COL2α was positive. Compared with the control group, the chondrocyte viability, apoptosis rate, ROS fluorescence intensity, p-p38-MAPK/p38-MAPK, and the expression of Bax protein in H2O22 group increased, the level of Col2α1 mRNA, and the expression of Col2α1 and Bcl-2 proteins decreased (P<0.05). Compared with H₂O₂ group, the chondrocyte viability, apoptosis rate, ROS fluorescence intensity, p-p38-MAPK/p38-MAPK, and the expression of Bax protein in dihydromyricetin low concentration group, dihydromyricetin high concentration group, and NAC group decreased, the level of Col2α1 mRNA, and the expression of Col2α1 and Bcl-2 proteins increased (P<0.05). CONCLUSION Dihydromyricetin may inhibit chondrocyte apoptosis, inflammatory reaction and oxidative stress by inhibiting ROS/p38-MAPK pathway. Dihydromyricetin may be a potential drug for treating osteoarthritis.
Animals ; Chondrocytes/metabolism* ; Apoptosis/drug effects* ; Hydrogen Peroxide/toxicity* ; Osteoarthritis/physiopathology* ; Mice, Inbred C57BL ; Reactive Oxygen Species/metabolism* ; Mice ; Flavonols/pharmacology* ; p38 Mitogen-Activated Protein Kinases/genetics* ; Cell Proliferation/drug effects* ; Male ; Signal Transduction/drug effects* ; MAP Kinase Signaling System/drug effects* ; Cells, Cultured

Objective To study the impacts of curcumin on the proliferation, migration and cisplatin (DDP) resistance of bladder cancer cells by regulating the liver kinase B1-AMP activated protein kinase-microtubule-associated protein 1 light chain 3 (LKB1-AMPK-LC3) signaling pathway. Methods Human bladder cancer cell line T24 was cultured in vitro, and its DDP resistant T24/DDP cells were induced by cisplatin (DDP). After treating T24 and T24/DDP cells with different concentrations of curcumin, the optimal concentration of curcumin was screened by MTT assay. T24 cells were randomly grouped into control group, curcumin group, metformin group, and combination group of curcumin and metformin. After treatment with curcumin and LKB1-AMPK activator metformin, the proliferation, autophagy, migration, and apoptosis of T24 cells in each group were detected by MTT assay, monodansylcadavrine (MDC) fluorescence staining, cell scratch assay, and flow cytometry, respectively. Western blot was used to detect the expression of proteins related to LKB1-AMPK-LC3 signaling pathway in T24 cells of each group. T24/DDP cells were randomly assigned into control group, curcumin group, metformin group, and combination group of curcumin and metformin. Cells were treated with curcumin and metformin according to grouping and treated with different concentrations of DDP simultaneously. Then, the effect of curcumin on the DDP resistance coefficient of T24/DDP cells was detected by MTT assay. T24/DDP cells were randomly grouped into control group, DDP group, combination groups of DDP and curcumin, DDP and metformin, DDP, curcumin and metformi. After treatment with DDP, curcumin, and metformin, the proliferation, autophagy, migration, apoptosis, drug resistance, and the expression of proteins related to LKB1-AMPK-LC3 signaling pathway in T24/DDP cells of each group were detected with the same methods. Results Compared with the control group, the activity of T24 cells, relative number of autophagosomes, migration rate, Phosphorylated-LKB1 (p-LKB1)/LKB1, Phosphorylated-AMPK (p-AMPK)/AMPK, LC3II/LC3I, and the DDP resistance coefficient of T24/DDP cells in the curcumin group were lower, and the apoptosis rate of T24 cells was higher; the changes in various indicators in the metformin group were opposite to those in the curcumin group. Compared with the curcumin group, the activity of T24 cells, relative number of autophagosomes, migration rate, p-LKB1/LKB1, p-AMPK/AMPK, LC3II/LC3I, and the DDP resistance coefficient of T24/DDP cells in the combination group of curcumin and metformin were higher, and the apoptosis rate of T24 cells was lower. Compared with the control group, there were no obvious changes in various indicators of T24/DDP cells in the DDP group. Compared with the control group and DDP group, the viability of T24/DDP cells, relative number of autophagosomes, migration rate, P-glycoprotein (P-gp) protein expression, p-LKB1/LKB1, p-AMPK/AMPK, and LC3II/LC3I in the combination group of DDP and curcumin were lower, and the apoptosis rate of T24/DDP cells was higher; the changes in the above indicators in the combination group of DDP and metformin were opposite to those in the combination group of DDP and curcumin. Compared with the combination group of DDP and curcumin, the viability of T24/DDP cells, relative number of autophagosomes, migration rate, P-gp protein expression, p-LKB1/LKB1, p-AMPK/AMPK, and LC3II/LC3I in the combination group of DDP, curcumin and metformin were higher, and the apoptosis rate of T24/DDP cells was lower. Conclusion Curcumin can reduce the activity of LKB1-AMPK-LC3 signaling pathway, thereby inhibiting autophagy, proliferation and migration of bladder cancer cells, promoting their apoptosis, and weakening their resistance to DDP.
Humans ; Cisplatin/pharmacology* ; Curcumin/pharmacology* ; Cell Proliferation/drug effects* ; Signal Transduction/drug effects* ; Protein Serine-Threonine Kinases/genetics* ; AMP-Activated Protein Kinases/metabolism* ; Drug Resistance, Neoplasm/drug effects* ; Urinary Bladder Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/drug effects* ; AMP-Activated Protein Kinase Kinases ; Microtubule-Associated Proteins/metabolism* ; Apoptosis/drug effects* ; Antineoplastic Agents/pharmacology* ; Metformin/pharmacology* ; Autophagy/drug effects*

Objective The aim of this study was to investigate the effect of p38 on stem cell maintenance of pancreatic cancer. Methods Human pancreatic cancer cells PANC-1 were treated with different concentrations of 5-fluorouracil(5-FU)(0.5×IC₅₀, IC₅₀, and 2×IC₅₀) for 24 hours, and VX-702 (p38 phosphorylation inhibitor) was added, and the cells were inoculated in 6-well culture dishes with ultra-low adhesion to observe the changes of sphere tumors. The expression levels of cyclin-dependent kinase 2(CDK2), cyclin B1 and D1, Octamer-binding transcription factor 4(OCT4), SRY-box transcription factor 2(SOX2), Nanog and p38 were measured by Western blot. The mRNA expression levels of p38, OCT4, Nanog and SOX2 were tested by RT-PCR. Cell cycle, apoptosis, and the proportion of CD44⁺CD133⁺PANC-1 cells were evaluated by flow cytometry. Results The results showed that 5-FU inhibited the formation of tumor spheres in PANC-1 cells, increased CD44⁺CD133⁺cell fragments, down-regulated the expression of OCT4, Nanog and SOX2, and inhibited the stemness maintenance of PANC-1 tumor stem cells. Phosphorylation of PANC-1 cells was inhibited by a highly selective p38 MAPK inhibitor, VX-702(p38 mitogen-activated protein kinase inhibitor), which had the same effect as 5-FU treatment. When VX-702 combined with 5-FU was used to treat PANC-1 cells, the therapeutic effect was enhanced. Conclusion p38 inhibitors decreased PANC-1 cell activity and increased cell apoptosis. p38 inhibitors inhibit the stemness maintenance of pancreatic cancer stem cells.
Humans ; Phosphorylation/drug effects* ; Cell Line, Tumor ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors* ; Neoplastic Stem Cells/metabolism* ; Drug Resistance, Neoplasm/drug effects* ; Fluorouracil/pharmacology* ; Pancreatic Neoplasms/pathology* ; Apoptosis/drug effects* ; SOXB1 Transcription Factors/genetics* ; Octamer Transcription Factor-3/genetics*

Objective To investigate the impact of Astragaloside-IV (AS-IV) on the progression of myocardial infarction (MI) through macrophage-dependent mechanisms by regulating Snail1 lactylation and acetylation, as well as the transforming growth factor β (TGF-β) pathway. Methods Oxygen glucose deprivation (OGD) was used to establish an in vitro myocardial ischemia model in rat cardiomyocytes (H9c2), which were then treated with AS-IV. Cell viability was assessed using CCK-8, apoptosis was evaluated by flow cytometry, and LDH levels were measured to assess cellular damage. RAW246.7 cells were treated with LPS, and lactate levels in the supernatant were measured using ELISA, while expression of macrophage phenotype markers was evaluated using Western blot. RAW246.7 cell-conditioned medium (CM) was co-cultured with H9c2 cells to assess the protective effects of AS-IV on macrophage CM-mediated H9c2 damage. RAW246.7 cells were induced to differentiate into M1-like macrophages using LPS (100 ng/mL) + IFN-γ (20 ng/mL), and Snail1 was overexpressed in M1 macrophages. Transfected M1 macrophage CM was co-cultured with H9c2 cells to validate the mechanisms of AS-IV in MI. An MI rat model was established by ligation of the left anterior descending coronary artery (LAD), and was treated with AS-IV. Cardiac function, myocardial cell apoptosis, and cardiac tissue pathology were studied using echocardiography, TUNEL, and HE staining, respectively. Results Compared to the OGD group, AS-IV treatment promoted cell viability, reduced apoptosis and decreased LDH release. LPS upregulated lactate levels in the supernatant of RAW246.7 cell cultures and induced polarization of RAW246.7 cells to the M1 phenotype. AS-IV attenuated the damaging effects of RAW246.7 cell CM on H9c2 cells . Overexpression of Snail1 in M1 macrophages weakened the protective effects of AS-IV on H9c2 cells . In vivo study, results showed that, compared to the MI group, AS-IV treatment reduced lactate levels in the hearts of MI rats, improved cardiac function and myocardial injury and attenuated myocardial cell apoptosis. Conclusion AS-IV inhibits TGF-β pathway activation through the suppression of Snail1 lactylation and acetylation in a macrophage-dependent manner, thereby mitigating myocardial cell damage following MI.
Animals ; Myocardial Infarction/drug therapy* ; Rats ; Snail Family Transcription Factors/metabolism* ; Macrophages/cytology* ; Myocytes, Cardiac/metabolism* ; Triterpenes/pharmacology* ; Saponins/pharmacology* ; Acetylation/drug effects* ; Apoptosis/drug effects* ; Mice ; Cell Line ; RAW 264.7 Cells ; Transforming Growth Factor beta/metabolism*

Objective To explore the characteristics of the inhibitory effect of Evodiamine on the proliferation of activated T cells. Methods Mononuclear cells from peripheral blood (PBMCs) were obtained from healthy donors through density gradient centrifugation, and T cells were subsequently purified by using immunomagnetic bead separation. T cell activation was induced by employing anti-human CD3 and anti-human CD28 antibodies. T cells were treated with different concentrations of EVO (0.37, 1.11, 3.33, and 10)μmol/L. Flow cytometry was applied to evaluate the proliferation index, apoptosis rate, viability, CD25 expression levels, and cell cycle distribution of T cells. The expression levels of cytokines IL-2, IL-17A, IL-4, and IL-10 were quantified by using ELISA. Results 1.11, 3.33 and 10 μmol/L EVO effectively inhibited the proliferation of activated T cells, with an IC₅₀ of (1.5±0.3)μmol/L. EVO did not induce apoptosis in activated T cells and affect the survival rate of resting T cells. EVO did not affect the expression of CD25 and the secretion of IL-2 in activated T cells. EVO arrested the T cell cycle at the G2/M phase, resulting in an increase in G2/M phase cells, and exhibited a concentration-dependent effect. EVO did not affect the secretion of IL-4, IL-10 by activated T cells, but significantly inhibited the secretion of IL-17A. Conclusion EVO did not significantly affect the activation process of T cells but inhibited T cell proliferation by arresting the cell cycle at the G2/M phase and significantly suppressed the secretion of the pro-inflammatory cytokine IL-17A, which suggests that EVO has the potential to serve as a lead compound for the development of low-toxicity and high-efficiency immunosuppressants and elucidates the mechanisms underlying the anti-inflammatory and immunomodulatory effects of the traditional Chinese medicine Evodia rutaecarpa.
Humans ; Cell Proliferation/drug effects* ; Quinazolines/pharmacology* ; T-Lymphocytes/metabolism* ; Lymphocyte Activation/drug effects* ; Apoptosis/drug effects* ; Interleukin-4/metabolism* ; Interleukin-10/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-17/metabolism* ; Interleukin-2/metabolism* ; Cell Cycle/drug effects* ; Cells, Cultured

Objective To investigate the effect and mechanism of Efferocytosis Relatived LncRNA (EFRL) on homocysteine-induced atherosclerosis in macrophage efferocytosis. Methods RAW264.7 cells were cultured in vitro, and the Control group (0 μmol/L Hcy) and Hcy intervention group (100 μmol/L Hcy) were set up. After GapmeR transfection of macrophages with Hcy intervention, EFRL knockdown negative control group (Hcy combined with LNA-NC) and EFRL knockdown group (Hcy combined with LNA-EFRL) were set up. High-throughput sequencing was applied for different expression of LncRNA MSTRG. 88917.16 (EFRL), UCSC was used to analyze its conservation, CPC and CPAT were used to analyze its ability to encode proteins, and GO and KEGG were used to analyze related biological functions. The localization of LncRNA EFRL in macrophages was analyzed by nucleoplasmic separation and RNA-FISH. Quantitative real-time PCR was used to detect the expression levels of LncRNA EFRL and its target gene SPAST in Hcy-treated macrophages. The apoptosis rate of Jurkat cells induced by UV was detected by flow cytometry. In vitro efferocytosis assay combined with immunofluorescence technique was used to analyze macrophage efferocytosis. ELISA was used to detect the levels of interleukin 1β(IL-1β) and IL-18. Results The new LncRNA MSTRG.88917.16 was identified and named EFRL(Efferocytosis Relatived LncRNA). UCSC, CPC and CPAT analyses showed that LncEFRL is highly conserved and does not have the ability to encode proteins. GO and KEGG analyses suggested that LncEFRL may be involved in macrophage efferocytosis. LncRNA EFRL was localized in the nucleus of macrophages as determined by nucleoplasmic separation and RNA-FISH. In comparison to the Control group, the expression levels of LncRNA EFRL and its target gene SPAST in the Hcy group were increased. In comparison to the Control group (0 min), the apoptosis rate of the experimental group (15, 30 min) Annexin V is more than 85%. Compared with Hcy combined with LNA-NC group, Hcy combined with LNA-EFRL group had enhanced macrophage efferocytosis and reduced levels of inflammatory factors. Compared with Hcy combined with LNA-NC group, the expression level of SPAST in Hcy combined with LNA-EFRL group was decreased. Conclusion Inhibition of EFRL expression can alleviate the process of Hcy inhibiting macrophage efferocytosis, and the mechanism is related to the regulation of the downstream target gene SPAST by EFRL.
RNA, Long Noncoding/physiology* ; Animals ; Homocysteine ; Mice ; Macrophages/drug effects* ; Humans ; RAW 264.7 Cells ; Atherosclerosis/chemically induced* ; Apoptosis/genetics* ; Phagocytosis/genetics* ; Jurkat Cells ; Interleukin-1beta/genetics* ; Efferocytosis

Objective To investigate the effect of gentiopicroside on osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs), and to determine whether its mechanism involves the stromal cell-derived factor 1(SDF-1)/C-X-C chemokine receptor 4 (CXCR4) pathway. Methods BMSCs were divided into six groups: normal culture control group, osteogenic induction model group, low-dose gentiopicroside (L-gentiopicroside, 10 μmol/L) group, medium-dose gentiopicroside (M-gentiopicroside, 20 μmol/L) group, high-dose gentiopicroside (H-gentiopicroside, 40 μmol/L) group, and H-gentiopicroside+SDF-1/CXCR4 pathway inhibitor (AMD3100) group (H-gentiopicroside+AMD3100, 40 μmol/L gentiopicroside+10 μg/mL AMD3100). Cell viability, apoptosis, ALP activity, mineralized nodule formation, and protein levels of the SDF-1/CXCR4 pathway were assessed using the CCK-8 assay, flow cytometry, ALP staining, Alizarin Red S staining, and Western blotting, respectively. Results No mineralized nodules were observed in either the control and model group, although the color of the model group deepened. Compared with the control group, the model group showed significantly increased A value, ALP activity, expression levels of Runt related transcription factor 2 (RUNX2), osteopontin (OPN), SDF-1, CXCR4 proteins, along with a lower apoptosis rate. Compared with the model group, the L-gentiopicroside, M-gentiopicroside and H-gentiopicroside groups showed dose-dependently (Lapoptosis rates. However, compared with the H-gentiopicroside group, the H-gentiopicroside+AMD3100 group showed reduced A value, ALP activity, expression levels of RUNX2, OPN, SDF-1, and CXCR4, along with a higher apoptosis rate. Conclusion The promoting effect of gentiopicroside on osteogenic differentiation of BMSCs may be achieved by activating the SDF-1/CXCR4 signaling pathway.
Humans ; Receptors, CXCR4/genetics* ; Mesenchymal Stem Cells/metabolism* ; Chemokine CXCL12/genetics* ; Iridoid Glucosides/pharmacology* ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Signal Transduction/drug effects* ; Cells, Cultured ; Apoptosis/drug effects* ; Bone Marrow Cells/metabolism*

PURPOSE: The secondary damage of spinal cord injury (SCI) starts from the collapse of the blood spinal cord barrier (BSCB) to chronic and devastating neurological deficits. Thereby, the retention of the integrity and permeability of BSCB is well-recognized as one of the major therapies to promote functional recovery after SCI. Previous studies have demonstrated that activation of hypoxia inducible factor-1α (HIF-1α) provides anti-apoptosis and neuroprotection in SCI. Endogenous HIF-1α, rapidly degraded by prolylhydroxylase, is insufficient for promoting functional recovery. Dimethyloxalylglycine (DMOG), a highly selective inhibitor of prolylhydroxylase, has been reported to have a positive effect on axon regeneration. However, the roles and underlying mechanisms of DMOG in BSCB restoration remain unclear. Herein, we aim to investigate pathological changes of BSCB restoration in rats with SCI treated by DOMG and evaluate the therapeutic effects of DMOG. METHODS: The work was performed from 2022 to 2023. In this study, Allen's impact model and human umbilical vein endothelial cells were employed to explore the mechanism of DMOG. In the phenotypic validation experiment, the rats were randomly divided into 3 groups: sham group, SCI group, and SCI + DMOG group (10 rats for each). Histological analysis via Nissl staining, Basso-Beattie-Bresnahan scale, and footprint analysis was used to evaluate the functional recovery after SCI. Western blotting, TUNEL assay, and immunofluorescence staining were employed to exhibit levels of tight junction and adhesion junction of BSCB, HIF-1α, cell apoptosis, and endoplasmic reticulum (ER) stress. The one-way ANOVA test was used for statistical analysis. The difference was considered statistically significant at p < 0.05. RESULTS: In this study, we observed the expression of HIF-1α reduced in the SCI model. DMOG treatment remarkably augmented HIF-1α level, alleviated endothelial cells apoptosis and disruption of BSCB, and enhanced functional recovery post-SCI. Besides, the administration of DMOG offset the activation of ER stress induced by SCI, but this phenomenon was blocked by tunicamycin (an ER stress activator). Finally, we disclosed that DMOG maintained the integrity and permeability of BSCB by inhibiting ER stress, and inhibition of HIF-1α erased the protection from DMOG. CONCLUSIONS Our findings illustrate that the administration of DMOG alleviates the devastation of BSCB and HIF-1α-induced inhibition of ER stress.
Spinal Cord Injuries/pathology* ; Animals ; Apoptosis/drug effects* ; Amino Acids, Dicarboxylic/therapeutic use* ; Recovery of Function/drug effects* ; Rats ; Rats, Sprague-Dawley ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Male ; Spinal Cord/blood supply*