Advances in the study on cytokines related to dental pulp regeneration / 口腔医学

@#With the development of molecular biology, biomaterials and tissue engineering, regenerative treatment of pulpal and periradicular diseases is facing new opportunities. At present, a large number of studies on dental pulp regeneration reveal that cytokines are essential for promoting migration, proliferation and osteogenic differentiation of dental pulp stem cells. In this paper, we review several kinds of cytokines related to dental pulp regeneration, and analyze their roles and regulatory mechanisms in dental pulp regeneration.

An experimental study on the biological characteristics of SDF-1/CS/β-GP composite bio-membrane in vitro / 口腔医学

Objective@#To prepare a composite membrane by chitosan/β-sodium glycerophosphate(CS/β-GP) thermosensitive hydrogel combined with stromal cell derived factor-1(SDF-1) and observe its biological characteristics in vitro.@*Methods@#Different doses of SDF-1 were added into CS/β-GP solution and then the thermosensitive gel time was measured. The SDF-1/CS/β-GP solution was membrane paved and dried to prepare composite membranes. The morphological characteristics were observed by scanning electron microscope(SEM). Composite membranes were placed into cell culture medium, and the supernatant(n=3) was extracted after standing at 6, 12, 24, 36, 48, 60 h, respectively. The concentration of SDF-1 in the solution was measured. Bone mesenchymal stem cells(BMSCs) were cultured in the Transwell room, and the composite membranes containing different concentrations of SDF-1 were placed in the lower chamber. There were four groups(n=3): Group M0 used CS/β-GP membrane(control group), Group M1, M2, M3 used SDF-1/CS/β-GP membrane(SDF-1 was 100, 200, 400 ng/mL respectively). After culture for 6, 12 and 24 h, the cells under the membrane were preserved and Giemsa stained and counted. The absorbance(OD) value was measured by MTT method to calculate the cell proliferation rate. SPSS 19.0 was used for multi-factor analysis of variance.@*Results @#After adding a certain amount of SDF-1 into CS/β-GP solution, the gel time did not change significantly(P>0.05). The SDF-1/CS/β-GP membrane was translucent and porous at 37 ℃. In this experiment, the volumic mass of SDF-1 released by SDF-1/CS/β-GP composite membrane increased gradually with the experimental time(P<0.01). Transwell cell chemotaxis test showed that the number of BMSCs cells with directional migration increased with the prolongation of observation time(P<0.01) and the increase of SDF-1 volumic mass(P<0.01). In MTT test, the OD value of migration cell solution increased with the prolongation of time(P<0.01) and the increase of SDF-1 volumic mass(P<0.01). @*Conclusion@# The SDF-1/CS/β-GP composite membrane has a porous structure and biological activity of chemotactic BMSCs directional migration. It is a potential membrane for guided tissue regeneration.

Scientific basis of acupuncture on mesenchymal stem cells for treating ischemic stroke / 中国针灸

The scientific basis of acupuncture on mesenchymal stem cells (MSCs) for treating ischemic stroke (IS) is discussed. MSCs transplantation has great potential for the treatment of tissue damage caused by early stage inflammatory cascade reactions of IS, but its actual transformation is limited by various factors. How to improve the homing efficiency of MSCs is the primary issue to enhance its efficacy. As such, the possible mechanisms of acupuncture and MSCs transplantation in inhibiting inflammatory cascade reactions induced by IS are explored by reviewing literature, and a hypothesis that acupuncture could promote the secretion of stromal cell-derived factor-1α (SDF-1α) from ischemic foci to regulate SDF-1α/CXC chemokine receptor 4 (CXCR4) axis, thereby improving the homing efficiency of MSCs transplantation, exerting its neuroprotective function, and improving the bed transformation ability, is proposed.

Bushen Jianpi Jiedu Liyan Formula Regulates Homing-associated Cell Adhesion Molecules in Rat Model of IgA Nephropathy / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Bushen Jianpi Jiedu Liyan formula on the expression of integrin alpha 4 beta 1 （α4 β1）， vascular cell adhesion molecule-1 （VCAM-1）， stromal-derived factor-1 （SDF-1）， and chemokine receptor-4 （CXCR4） in the small intestine and bone marrow of the rat model of immunoglobulin A（IgA） nephropathy. MethodA total of 120 male SD rats were used to establish the IgA nephropathy model by intragastric administration of bovine serum albumin （BSA）， subcutaneous injection of CCl4， and tail vein injection of lipopolysaccharide （LPS）. The successfully modeled rats were randomized into blank， model， lotensin （63 mg·kg-1）， and low-， medium-， and high-dose （10.4， 20.81， 41.62 g·kg-1， respectively） Bushen Jianpi Jiedu Liyan formula groups （n=16）. The rats were treated with corresponding drugs according to their body weight. After 7 weeks of administration， the rats were sacrificed for the collection of samples， and the protein and mRNA levels of α4 β1， VCAM-1， SDF-1， and CXCR4 in the small intestine and bone marrow were determined by immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction， respectively. ResultCompared with the blank group， the model group showed increased red blood cell count in the urine at the 10th， 12th， 14th， 16th weeks （P<0.01）， and such increases were reduced in the drug intervention groups （P<0.05）， especially in the medium-dose Bushen Jianpi Jiedu Liyan formula group （P<0.05）. Compared with those in the blank group， the protein levels of α4 β1， VCAM-1， SDF-1， and CXCR4 in the intestinal lamina propria in the model group were up-regulated （P<0.05）， and such un-regulations were inhibited in the drug intervention groups （P<0.05）. Compared with the model group， medium-dose Bushen Jianpi Jiedu Liyan formula down-regulated the protein levels of SDF-1 and CXCR4 in the intestinal lamina propria （P<0.05）. Compared with the blank group， the model group showed down-regulated mRNA levels of α4 β1 and SDF-1 and up-regulated mRNA levels of VCAM-1 and CXCR4 （P<0.05）. Compared with the model group， the drug intervention groups showed down-regulated mRNA levels of SDF-1 and CXCR4 （P<0.05）. ConclusionBushen Jianpi Jiedu Liyan formula regulates the expression of α4 β1， VCAM-1， SDF-1， and CXCR4 in the intestinal lamina propria to inhibit the homing effect of plasma cells， which may be associated with the Toll-like receptor-mediated activation of immune response. Bushen Jianpi Jiedu Liyan formula can down-regulate the expression of adhesion molecules to inhibit the proliferation of plasmocytes in circulation， so as to reduce the renal injury of IgA nephropathy.

Neotuberostemonine and tuberostemonine ameliorate pulmonary fibrosis through suppressing TGF-β and SDF-1 secreted by macrophages and fibroblasts via the PI3K-dependent AKT and ERK pathways / 中国天然药物

Activated fibroblasts and M2-polarized macrophages may contribute to the progression of pulmonary fibrosis by forming a positive feedback loop. This study was aimed to investigate whether fibroblasts and macrophages form this loop by secreting SDF-1 and TGF-β and the impacts of neotuberostemonine (NTS) and tuberostemonine (TS). Mice were intratracheally injected with 3 U·kg-1 bleomycin and orally administered with 30 mg·kg-1 NTS or TS. Primary pulmonary fibroblasts (PFBs) and MH-S cells (alveolar macrophages) were used in vitro. The animal experiments showed that NTS and TS improved fibrosis related indicators, inhibited fibroblast activation and macrophage M2 polarization, and reduced the levels of TGF-β and SDF-1 in alveolar lavage fluid. Cell experiments showed that TGF-β1 may activated fibroblasts into myofibroblasts secreting SDF-1 by activating the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways. It was also found for the first time that SDF-1 was able to directly polarize macrophages into M2 phenotype secreting TGF-β through the same pathways as mentioned above. Moreover, the results of the cell coculture confirmed that fibroblasts and macrophages actually developed a feedback loop to promote fibrosis, and the secretion of TGF-β and SDF-1 was crucial for maintaining this loop. NTS and TS may disturb this loop through inhibiting both the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways to improve pulmonary fibrosis. NTS and TS are stereoisomeric alkaloids with pyrrole[1,2-a]azapine skeleton, and their effect on improving pulmonary fibrosis may be largely attributed to their parent nucleus. Moreover, this study found that inhibition of both the AKT and ERK pathways is essential for maximizing the improvement of pulmonary fibrosis.

Effect of Astragali Radix-Curcumae Rhizoma on SDF-1/CXCR4/NF-κB Signaling Pathway of Orthotopic Transplantation Model of Colon Cancer in Mice / 中国实验方剂学杂志

Objective:To explore the possible mechanism of Astragali Radix-Curcumae Rhizoma （AC） in inhibiting tumor growth in the orthotopic transplantation model of colon cancer in mice. Method:The molecular docking technology was used to predict the intermolecular interaction between the main active components of AC and the pathway target proteins， such as stromal cell-derived factor-1 （SDF-1）， C-X-C motif chemokine receptor 4 （CXCR4）， and nuclear factor kappa-B p65 （NF-<italic>κ</italic>B p65）. The orthotopic transplantation model of CT26.WT colon cancer was established in mice for <italic>in vivo</italic> experimental verification. Sixty BALB/c male mice were randomly divided into a sham operation group， a model group， a 5-fluorouracil （5-Fu， 30 mg·kg^-1） group，and low- （0.32 g·kg^-1）， medium- （0.64 g·kg^-1）， and high-dose （1.28 g·kg^-1） AC groups， with 10 mice in each group. The sham operation group and the model group received normal saline by gavage. The corresponding drugs were administered by gavage in the 5-Fu group and by intraperitoneal injection in the AC groups. After intervention for 15 days， the tumor <italic>in situ</italic> was completely stripped， and the colon tissues 5-6 cm in length adjacent to the tumor were taken. The tumor volume was measured and calculated. The pathological changes of tumor tissues and colon tissues were observed by Hematoxylin-Eosin （HE） staining. Western blot was used to detect the protein expression of SDF-1， CXCR4， p-NF-<italic>κ</italic>B p65 in colon tissues. Western blot and Real-time quantitative polymerase chain reaction （Real-time PCR） were used to detect SDF-1， CXCR4， NF-<italic>κ</italic>B p65， Cyclin D₁， oncogene c-Myc protein and mRNA expression in tumor tissues. Result:Compared with the model group， 5-Fu and AC groups showed reduced tumor volumes <italic>in situ</italic> （<italic>P</italic><0.05， <italic>P</italic><0.01）， with the tumor inhibition rate in the 5-Fu group as high as （61.38±2.34）%. The tumor-inhibiting effect was optimal in the medium-dose AC group， with the tumor inhibition rate of （43.43±3.71）%. Compared with the model group， 5-Fu and AC groups showed relieved pathological changes of tumor and colon tissues. Specifically， AC down-regulated the protein expression levels of SDF-1， CXCR4， and p-NF-<italic>κ</italic>B p65 in colon tissues （<italic>P</italic><0.01）， and down-regulated the protein and mRNA expression levels of SDF-1， CXCR4， NF-<italic>κ</italic>B p65， Cyclin D₁， and c-Myc in tumor tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:AC can inhibit the growth of orthotopic transplantation tumor of colon cancer， and its intervention mechanism may be related to the regulation of related protein and mRNA expression in the SDF-1/CXCR4/NF-<italic>κ</italic>B signaling pathway.

Effect and Mechanism of Qixian Tongluo Prescription on Neural Function Recovery in Patients with Cerebral Infarction / 中国实验方剂学杂志

Objective:To investigate the effect of Qixian Tongluo prescription on neural function recovery in patients with cerebral infarction and its mechanism. Method:A total of 100 inpatients （January to June，2020）with cerebral infarction in the Neurology Department of Wenzhou Hospital of Traditional Chinese Medicine were assigned to an experimental group （<italic>n</italic>=50） and a control group （<italic>n</italic>=50） according to the random number table. Both groups received conventional treatment of western medicine，while the experimental group took additional Qixian Tongluo prescription. Treatment lasted for 12 weeks. The clinical efficacy，National Institutes of Health Stroke Scale （NIHSS） score， the modified Barthel index （MBI），Fugl-Meyer assessment （FMA） score， and levels of brain-derived neurotrophic factor（BDNF），vascular endothelial growth factor（VEGF）， and stromal cell-derived factor-1（SDF-1） in peripheral blood of the two groups before and after treatment were compared. Result:The total response rate in the experimental group was 84.00%（42/50），higher than 66.00%（33/50） in the control group （<italic>Z</italic>=-7.365，<italic>P</italic><0.05）. There was no significant difference in the scores of MBI，FMA， and NIHSS before treatment between the two groups. The MBI and FMA scores of the two groups increased （<italic>P</italic><0.01）， and the NIHSS scores decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the control group after treatment， the experimental group showed increased MBI and FMA scores and decreased NIHSS score （<italic>P</italic><0.05）. There was no significant difference in BDNF level between the two groups before and after treatment. The VEGF and SDF-1 levels in the peripheral blood of the two groups were higher than those before treatment （<italic>P</italic><0.05）， and the experimental group was higher than the control group （<italic>P</italic><0.05）. Conclusion:Qixian Tongluo prescription can effectively improve the clinical efficacy，the quality of life， and the prognosis of patients with cerebral infarction during convalescence. The underlying mechanism is associated with the promotion of the expression of endogenous VEGF and SDF-1 in the peripheral blood to activate the SDF-1/chemokine receptor 4（CXCR4） signaling pathway， induce the recruitment and mobilization of endothelial progenitor cells， and facilitate the angiogenesis and repair of ischemic brain tissues.

CXCR4/SDF-1 axis regulates the effect of human lung adenocarcinoma PC-9 cells on function of in vitro blood-brain barrier model formed by Bends cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the influences of human lung adenocarcinoma PC-9 cells on tight junction proteins of blood brain barrier (BBB) under CXCR4/SDF-1 axis by establishing a model of BBB in vitro. Methods: The immortalized mouse brain microvascular endothelial Bends cells were used to establish a model of BBB in vitro by monolayer culture; Subsequently, transendothelial electric resistance (TEER) and fluorescein sodium permeability experiment were used to detect the function of in vitro BBB model and observe the effect of PC-9 cells on the function of BBB model, respectively. Western blotting was used to detect the effect of PC-9 cells on function of BBB model and expressions of endothelial tight junction proteins under the treatment of single or combined AMD3100 and SDF-1 (1 μg/ml AMD3100,100 ng/ml SDF-1, AMD3100+SDF-1). Transwell assay was used to detect the influence of CXCR4/SDF-1 axis on the ability of PC-9 cells transmigrating the cell layer of BBB model. Results: Monolayer culture of Bends cells can form tightly connected BBB withhighTEER,which reached (182.13±5.19) Ω.cm2 at the 96 h; in the meanwhile, fluorescein sodium permeability experiment showed that BBB had significantly lower permeability than that of control group ([40.31±2.43]% vs [150.10±3.17]%, P<0.05). The TEER of BBB decreased to (46.7±4.35) Ω·cm2 after coculture with PC-9 cells for 24 h, and at the same time the sodium fluorescein permeability of BBB significantly increased than that of pre-treatment ([136.32±4.93]% vs [50.24±6.21]%, P<0.05). PC-9 cells up-regulated the expressions of tight junction proteins of Bends cells under the treatment of AMD3100 (P<0.05). The number of PC-9 cells transmigrating the BBB inAMD3100 treatment group was significantly lower than that of CON group (43±2 vs 81±2, P<0.05). Conclusion: AMD3100 can reduce the ability of PC-9 cells destroying the tight junction of the BBB model established in vitro by Bends cells.

Effect of Duhuo Xuduan Tang for Oral Administration and Iontophoresis on SDF-1/CXCR4 Signaling Pathway and Relevant Factors of Cartilage Degradation in Knee Joint Fluid of Patients with Knee Osteoarthritis with Liver and Kidney Deficiency / 中国实验方剂学杂志

Objective::To observe the clinical efficacy of Duhuo Xuduan Tang for oral administration and iontophoresis in the treatment of knee osteoarthritis (KOA) with liver and kidney deficiency and its effect on stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling pathway. Method::Totally 150 KOA patients with deficiency of liver and kidney diagnosed in the Teaching Hospital of Tianjin University of Traditional Chinese Medicine(TCM) were randomly divided into control group, oral TCM group and iontophoresis group, with 50 cases in each group. The control group was given glucosamine sulfate capsule, 0.5 g/time, twice a day, while the oral TCM group was given Duhuo Xuduan Tang, 150 mL/time, twice a day. In the iontophoresis group, Duhuo Xuduan Tang was administered at Kuangu acupoint, Xiguan acupoint, Xiyan acupoint and Dubi acupoint for iontophoresis for 30 minutes, once a day. All of the three groups were treated for 4 weeks. The swelling degree and the pain degree of knee joint before and after treatment were observed, and the clinical efficacy was recorded. The protein contents of SDF-1, CXCR4, matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13) in knee joint fluid before and after treatment were detected by enzyme-linked immunosorbent assay (ELISA). Result::The efficacy of oral TCM group was better than that of iontophoresis group and control group, and the recurrence rate was the lowest (P<0.05). Compared with before treatment, the tenderness increased, whereas visual analogue scale(VAS) score, knee swelling score, The Western Ontario and McMaste Universities (WOMAC) score and SDF-1, CXCR4, MMP-3 and MMP-13 protein content in knee joint fluid decreased in oral TCM group after treatment, which were better than those in iontophoresis group and control group (P<0.05). Conclusion::Duhuo Xuduan Tang for oral administration and iontophoresis has an obvious effect on KOA with liver and kidney deficiency, with the best effect through oral administration. Its mechanism may be related to the inhibition of SDF-1/CXCR4 inflammatory signaling pathway and cartilage decomposition.

Effect of Ru′ai Shuhou Prescription on Proliferation and Metastasis of Breast Cancer MDA-MB-453 Cells Through SDF-1/CXCR4 Biological Axis / 中国实验方剂学杂志

Objective:To investigate the effect of Ru′ai Shuhou prescription （RSR） drug-containing serum on the proliferation and invasion ability of breast cancer cells MDA-MB-453 based on the biological axis of stromal cell-derived factor-1（SDF-1）/chemokine receptor 4 （CXCR4）. Method:A model of MDA-MB-453 cells with SDF-1-induced high expression of CXCR4 was established， and the rat drug-serum containing RSR and blank rat serum were prepared respectively. The cells were divided into fetal bovine serum control group （Blank）， blank rat serum group， SDF-1+blank rat serum group， SDF-1+RSR group， AMD3100+ SDF-1+blank rat serum group， and AMD3100+ SDF-1+RSR group. After intervention for 48 h， cell proliferation was detected by cell counting kit-8 （CCK-8） assay， cell invasion ability was detected by transwell assay， and mRNA and protein expressions of CXCR4， matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Result:As compared with the blank serum group， the proliferation of MDA-MB-453 cells was promoted and expression of CXCR4 mRNA was increased significantly when SDF-1 was 100 μg·L-1 （P<0.05）. As compared with SDF-1+blank rat serum group， RSR inhibited the proliferation and invasion of MDA-MB-453 cells induced by SDF-1， and at the same time， down-regulated the mRNA and protein expressions of CXCR4， MMP-2 and MMP-9 （P<0.05）. After pre-treatment with AMD3100 for 24 h， the inhibitory effect of RSR to cell proliferation was significantly increased （P<0.05）， and meanwhile， the decreases in mRNA and protein expression of CXCR4， MMP-2 and MMP-9 were more obvious， with statistically significant differences （P<0.05）. Conclusion:Through SDF-1/CXCR4 biological axis， RSR could down-regulate the expression of MMP-2 and MMP-9， reduce the degradation of extracellular matrix （ECM）， and then inhibit the metastasis of MDA-MB-453 cells. In addition， it has a synergistic effect with CXCR4 inhibitor AMD3100.

Evaluation of SDF-1 complex with CGF applied to canine dental-pulp-like tissue regeneration / 上海交通大学学报（医学版）

Objective: To evaluate the feasibility of immature permanent teeth pulp regeneration with a new method that utilizes the integration of concentrated growth factor (CGF) as a scaffold and stromal cell-derived factor-1 (SDF-1). Methods: Canine dental pulp cells (DPC) were isolated and cultured in vitro. Then the effects of SDF-1 and CGF were observed on DPC proliferation and differentiation. The pulpless model was established on the beagle’s immature incisors which were divided into four groups: natural pulp (A), empty cannel (B), CGF-filling (C) and SDF-1/CGF-filling (D). After 10 weeks, specimens were checked by imaging examination, RT-PCR and histological observation. Results: CGF extraction (CGFe) induced DPC proliferation while the combination of SDF-1 and CGFe enhanced this effect and also facilitated odontogenic and angiogenic differentiation of DPC. According to imaging examination, the apex growth of all four groups was in varying degrees. RT-PCR indicated the expressions of odontogenesis and angiogenesis related genes in group D were higher than those in group C. Besides, neonatal dentin and dental-pulp-like tissue were observed inside the canal of both group C and D, while only cementum-like tissue existed around root apex of group B. Conclusion:SDF-1 plays an important role in driving the process of pulp-like-tissue regeneration of immature permanent teeth by using CGF as an effective scaffold.

Effects of herbal-cake-separated moxibustion on the repair of vascular endothelial and SDF-1 in rabbits with atherosclerosis / 中国针灸

OBJECTIVE@#To observe the effects of herbal-cake-separated moxibustion on the repair of damaged vascular endothelium structure and the content of stromal cells derived factor 1 (SDF-1) in rabbits with atherosclerosis.@*METHODS@#A total of 75 rabbits were randomly divided into a normal group, a model group, a direct moxibustion group, an atorvastatin calcium group and a herbal-cake-separated moxibustion group, 15 rabbits in each one. The rabbits in the normal group were fed with normal diet, and the remaining rabbits were fed with high-cholesterol diet for 12 weeks to prepare atherosclerotic model. Two groups of acupoints, one was "Juque" (CV 14), "Tianshu" (ST 25) and "Fenglong" (ST 40), the other one was "Xinshu" (BL 15), "Ganshu" (BL 18) and "Pishu" (BL 20), were applied in the direct moxibustion group and herbal-cake-separated moxibustion group; the two groups of acupoints were selected alternatively every other day. The moxibustion was given for 30 min per treatment, once a day for 4 weeks. The rabbits in the atorvastatin calcium group were treated with atorvastatin calcium tablets (1.96 mg•kg•d) which were crushed into powder and mixed into breakfast. After modeling, the rabbits in the normal group and model group received no treatment, and immobilized at the time when moxibustion was applied in other three groups. The levels of total cholesterol (TC) and triglyceride (TG) were measured by enzymic method; the low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by colorimetric method; the morphological structure of aortic wall was observed under optical microscope; the serum level of SDF-1 was determined by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#After treatment, compared with the normal group, the levels of TC, TG and LDL-C were significantly increased in the model group (all <0.01), and the level of HDL-C was decreased (<0.01). Compared with the model group, the levels of TC, TG and LDL-C were significantly decreased (all <0.01), and the level of HDL-C was significantly increased in the direct moxibustion group, atorvastatin calcium group and herbal-cake-separated moxibustion group (<0.01, <0.05). Compared with the normal group, the morphological structure of aortic wall was significantly damaged in the model group. Compared with the model group, the vascular endothelial structure was improved in the atorvastatin calcium group and herbal-cake-separated moxibustion group, and the pathological change of aorta endothelial in the direct moxibustion group was relieved. After treatment, compared with the model group, the level of SDF-1 was increased in the direct moxibustion group, atorvastatin calcium group and herbal-cake-separated moxibustion group (<0.05, <0.01); the level of SDF-1 in the herbal-cake- separated moxibustion group was higher than that in the direct moxibustion group (<0.05).@*CONCLUSION@#The herbal- cake-separated moxibustion can promote the expression of SDF-1 in serum and repair the damaged aortic endothelial structure.

Effect of triptolide in improving platelet activation in patients with ankylosing spondylitis by regulating VEGFA,SDF-1,CXCR4 pathway / 中国中药杂志

The effect of triptolide( TP) on VEGFA,SDF-1,CXCR4 pathway were investigated in vitro to explore the mechanism in improving platelet activation in patients with ankylosing spondylitis( AS). Peripheral blood mononuclear cells( PBMC) were used for the experiment and divided into 4 groups: normal group( NC),model group( MC),triptolide group( TP),and AMD3100 group. The optimal concentration of TP was measured by the MTT method. The expressions of TNF-α,IL-1β,IL-4,IL-10,VEGFA and VEGFR were detected by ELISA. The expressions of SDF-1,CXCR4 and VEGFA were detected by real-time quantitative PCR( RT-qPCR).The expressions of SDF-1,CXCR4,VEGFA and VEGFR were detected by Western blot. The expression levels of CD62 p,CD40 L and PDGFA were detected by immunofluorescence. MTT results showed that medium-dose TP had the strongest inhibitory effect on cells at24 h. The results of ELISA and PCR showed that TP inhibited mRNA expressions of IL-1β,TNF-α,VEGFA,VEGFR and SDF-1,CXCR4 and VEGFA. The results of Western blot indicated that TP inhibited SDF-1,CXCR4 and VEGFA,VEGFR protein expressions; immunofluorescence results indicate that TP can inhibit the expressions of CD62 p,CD40 L,PDGFA. TP may regulate platelet activation by down-regulating SDF-1,CXCR4,VEGFA and VEGFR mRNA expressions,thereby down-regulating IL-1β and TNF-αexpressions,and up-regulating the expressions of IL-4 and IL-10 cytokines.

SDF-1α/CXCR4 axis promotes migration and invasion of pancreatic cancer cells through inducing epithelial-mesenchymal transition / 中国病理生理杂志

AIM:To investigate the role of SDF-1α/CXCR4 axis in pancreatic cancer cell migration and invasion.METHODS:The mRNA expression of CXCR4 in 4 pancreatic cancer cell lines was detected by RT-qPCR.The migration and invasion abilities of PANC-1 cells with the axis activated by exogenous SDF-1αor inhibited by CXCR4 inhibitor AMD3100 were detected by Transwell assays.The cell viability was measured by MTS assay.The protein expression of the epithelial-mesenchymal transition (EMT) -related molecules in the cells treated with exogenous SDF-1αor AMD3100 was determined by Western blot.RESULTS:All of the 4 pancreatic cancer cell lines expressed CXCR4 mRNA, while the PANC-1 cell line expressed the most.Exogenous SDF-1αpromoted the migration and invasion abilities of PANC-1 cells, which was inhibited by AMD3100.The PANC-1 cells treated with exogenous SDF-1αfor 72 h grew faster, while SDF-1αcombined with AMD3100 made little significance to the viability of PANC-1 cells.Exogenous SDF-1αinduced EMT of PANC-1 cells by up-regulating the expression of SNAIL and TWIST, and AMD3100 reversed this effect.CONCLUSION:SDF-1α/CXCR4 axis enhances the migration and invasion abilities of pancreatic cancer cells through inducing EMT.

Roles of integrin αvβ3 in SDF-1/CXCR4-induced choroidal neovascularization / 眼科新进展

Objective To explore the role of integrin αvβ3 in the promotion of the development of choroidal neovascularization (CNV) by SDF-1/CXCR4.Methods This study was divided into two parts in vitro and in vivo.As for the in vivo study,a CNV model was induced by laser on C57BL/6J mice,and then assigned into 4 groups:mice with solely CNV modeling as control group,with intravitreal injection of SDF-1 after immediate CNV modeling as SDF-1 group,with intravitreal injection of SDF-1 + CXCR4 inhibitor (AMD3100) after CNV modeling as SDF-1 + AMD3100 group,and mice with intravitreal injection of SDF-1 + αvβ3 inhibitor (SB273005) after modeling as SDF-1 + SB273005 group.CXCR4 and αvβ3 expression levels in laser-induced eyes were quantified by qRT-PCR at time points of day 1,3,5,7,10 and 14 after modeling,and immunofluorescence staining was applied to detect αvβ3 expression in regional CNV and its endothelial cells in the four groups.Finally,OCT was used to observe the height of retinal pigment epithelial (RPE) layers in CNV after treatment in the four groups.Moreover,in the experiment in vitro,Western blot was used to measure the expression of CXCR4 protein of RF/6A cells in normal control group,Si-CXCR4 knockdown group and Si-NC knockdown model group.Meanwhile,the expression of integrin subunit β3 protein was determined in the normal control group,SDF-1 group,SDF-1 + AMD3100group,SDF-1 + Si-NC group and SDF-1 + Si-CXCR4 group.Transwell assay was conducted to detect the migration ability of RF/6A cells in the normal control group,SDF-1group,SDF-1 +AMD3100 group,SDF-1 + SB273005 group.Results On the one hand,the study in vivo,qRT-PCR showed that the expression of CXCR4 and integrin subunit β3 mRNA was up-regulated at first,and then down-regulated with time passed after CNV induction,with the highest expression level of CXCR4 mRNA (4.263 ± 0.464) on day 3,and the peak expression of β3 mRNA (3.678 ±0.364) on day 7 after CNV modeling.The results of immunofluorescence staining showed that the β3 fluorescence intensity of SDF-1 group was significantly enhanced,and the ratio of β3/CD31 was also significantly increased,which both were significantly higher than those of the control group (P ＜ 0.01).However,the β3 fluorescence intensity and β3/CD31 ratio of SDF-1 +AMD3100 group and SDF-1 + SB273005 group were significantly weakened and decreased,respectively (P ＜0.05).OCT showed that the elevation level of RPE layer inSDF-1 group was significantly higher than that in the control group [(135.503 ± 10.301) μm vs.(94.443 ± 12.156) μm](P＜0.05).The height of RPE uplift in SDF-1 + AMD3100 group [(95.283 ±20.062) μm] and SDF-1 + SB273005 group [(99.807 ± 10.403) μm] was significantly decreased (P ＜ 0.05).On the other hand,in experiment in vitro,Western blot showed that the expression levels of integrin β3 in SDF-1 group and SDF-1 + Si-NC group were significantly higher than those in the control group [(1.301 ± 0.043) and (1.273 ± 0.077) vs.(0.244 ± 0.069)] (P ＜ 0.01).The levels of integrin subunit β3 protein in SDF-1 + si-CXCR4 group (0.322 ± 0.042) and SDF-1 + AMD3100 group (0.336 ± 0.077) were significantly down-regulated (P ＜ 0.01).Transwell assay showed that the amount of migrating cells in SDF-1 group increased,which was significantly higher than that of the control group (P ＜ 0.01),while the number of migrating cells in SDF-1 +AMD3100 group and SDF-1 + SB273005 group was significantly decreased.Conclusion Integrin αvβ3 can promote the development of CNV by mediating SDF-1/CXCR4 signaling in endothelial cells.

Establishment and Application of High-throughput Screening Model for Active Ingredients of Traditional Chinese Medicine Based on CXCR4 Promoter / 中国中医药信息杂志

Objective To establish a cell model targeting the CXCR4 gene promoter to screen of drugs potentially promoting the stem cell homing, and to screen the monomers of traditional Chinese medicine. Methods The human CXCR4 gene promoter sequence (279 bp) was inserted into the luciferase reporter vector pGL4.17-Basic to construct the recombinant plasmid pGL4.17-CXCR4, which was co-transfected with the internal reference plasmid pRL-TK into 293 cells. After verifying transcriptional activity of CXCR4 promoter, single cell clones were cultured to obtain a stable transfectant cell line and used for screening of candidate Chinese herbal monomer (1 to 10). Finally, Western blot was used to verify the effect of the selected Chinese herbal monomer on the expression of CXCR4 in MSCs. Results The drug screening cell model was successfully established and the candidate monomer 6 was screened out because of the strongest effect on enhancing CXCR4 promoter activity. Western blot results verified that the candidate monomer 6 could promoted the expression of CXCR4 of MSCs. Conclusion The drug screening cell model established in this study was able to achieve high-throughput screening for potentially promoting the stem cell homing Chinese herbal monomer.

Mechanism of Duhuo Jisheng decotion in delaying degeneration of nucleus pulposus cells in human intervertebral disc / 中国中药杂志

This paper aimed to investigate the role of Duhuo Jisheng decotion (DHJSD) in delaying human disc degeneration and its possible molecular mechanism. The intervertebral disc specimens were divided into normal and degenerated groups according to Pfirrmann classfication. The expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in intervertebral disc tissue were detected by Western blot and PCR. Then degenerated human primary NPCs were cultured in vitro, the viability of NPCs treated with stromal cell-derived factor-1 (SDF-1，10 μg·L⁻¹)and various concentrations of DHJSD was assessed by the CCK-8 assay, and the appropriate concentration was screened. The experiment was divided into three groups, control group, SDF-1 group and DHJSD plus SDF-1 group. The levels of TNF-α, IL-1β, Agg, coIⅡ, MMP-3 and MMP-13 were detected. The levels of CXCR4, NF-κB major groups P65 phosphorylation (p-P65) and nuclear translocation, after treated with CXCR4 siRNA and NF-κB inhibitor (BAY11-7082) were measured by Western blot and immunofluorescence. At the same time, the expression of cell inflammatory factors and extracellular matrix were also measured. The expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in the degenerated intervertebral disc tissue were significantly increased. In vitro study, the results of CCK-8 indicated that the viability of NPCs was significantly increased when DHJSD concentration was 300 mg·L⁻¹. After the experiment was divided into three groups, compared with SDF-1 group, the expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in DHJSD group were significantly decreased, but the expressions of Agg, coIⅡ were significantly increased. When CXCR4-siRNA was transfected into NPCs, SDF-1 increased expressions of CXCR4 and p-P65 and inhibited nuclear translocation of P65, whose effect was suppressed by CXCR4-siRNA and DHJSD. In addition, when BAY11-7082 was used to treat NPCs, the expression of TNF-α, IL-1β, MMP-3 and MMP-13 were significantly decreased. DHJSD could inhibit the production of inflammatory factors and promote the synthesis of extracellular matrix. The potential mechanism may be related to the SDF-1/CXCR4/NF-κB signaling pathway.

SDF-1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via NF-κB pathway / 基础医学与临床

Objective To investigate the role of SDF-1/CXCR4 axis on the apoptosis of human degenerative nucleus pulposus cells (NPCs) and its potential molecular mechanism .Methods The intervertebral disces tissues from clinical discectomy were divided into normal group and intervertebral disc degeneration ( IVD) group according to Pfirrmann classification.The different expression of SDF 1 and CXCR4 in human IVDs was tested by immunohistochemistry, quantify polymerase chain reaction (q-PCR) and Western blot.The primary degenerative NPCs were primary cultured.The generation Ⅲ~Ⅴ NPCs was treated with 10 ng/mL SDF-1, in the presence of or in the absence of CXCR4 siRNA transfection and 20 μmol/L NF-κB inhibitor (pyrrolidine dithiocar bamate,PDTC).The transfection efficiency and target protein of signal pathway were verified by Western blot , the apoptosis of NPCs were tested by Annexin V /PI, the nucleus transferlocation of P65 from NF-κB were tested by immunofluorescent method.Results SDF-1and CXCR4 were both expressed in all donor tissues, however, there was a significantly increased in the degenerative IVDs .The apoptosis of degenerative NPCs was expedited by SDF -1 stimulation,which was significantly suppressed by CXCR 4 silencing by siRNA (P<0.05).Furthermore, with SDF-1 stimulation,the expressions of phosphorylated P 65 was significantly increased and the P65 perssad transferred to the nucleuses,which could be suppressed by the NF-κB inhibitor, PDTC(P<0.05).Conclusions The expression levels of SDF-1 and CXCR4 are increased in degenerative NP tissue.The SDF-1/CXCR4 axis is considered to induce apoptotic of human degenerative NPCs via the NF-κB signaling pathway.

The influence of the transfection of SDF-1αto the heart mediated by recombinant adenovirus ,microbubbles and ultrasound on non targeted tissues of rats with acute myocardial infarction / 中华超声影像学杂志

Objective To evaluate gene transfection in liver , lung and kidney by ultrasound , microbubble and recombinant adenovirus mediated exogenous stromal cell derived factor-1α ( SDF-1α) gene transfer to the heart in rats with acute myocardial infarction( AMI) . Methods Forty AMI SD rats were randomly divided into control and experimental groups:myocardial infarction + ultrasound irradiation group (M+ U/control group , n = 10) ,and 3 experimental subgroups on the basis of pAd-EGFP/SDF-1α ( The biotin recombinant adenovirus expressing enhanced green fluorescent protein and SDF-1α) . Genes transfection length of time:1 day ,2 days and 3 days of transfection ( M +S1+U ,M+S2+U and M+S3+U group) . The expression of EGFP in liver ,lung and kidney were detected by laser scanning confocal microscopy at 7 days after administration . Results There was a little expression of EGFP in the liver ,lung and kidney in the drug delivery group and no expression in the control group .The differences in the expression of EGFP between all the gene delivery groups and the control group were statistically significant ( P <0 .05) . With the increase of the number of medication days , the target gene transfection increased slightly ,but there was no significant difference among the different drug delivery groups . Conclusions When the ultrasound ,microbubble and recombinant adenovirus mediated exogenous SDF-1αgene transfer to the heart in AMI rats ,liver ,lung and kidney tissues will also be transfected . However ,with the increasing of the days of administration , the transfection of target gene in non-target tissue produces only a slight accumulation . The transfection area of target gene in non-target tissue is not linear correlated with the days of administration .

Effect of SDF-1/CXCR7 on inflammatory cytokine synthesis and secretion in gastric cancer SGC-7901 cells / 西安交通大学学报(医学版)

ABSTRACT:Objective To investigate the effect of SDF-1/CXCR7 on inflammatory cytokine synthesis and secretion in gastric cancer SGC-7901 cells.Methods CXCR7 gene in SGC-7901 cells was silenced by shRNA lentiviral vector and the expression of CXCR7 was detected using Western blot and Real-time PCR.There were four groups as follows:LV-shRNA-NC,LV-shRNA-NC+SDF-1,LV-shRNA-CXCR7,and LV-shRNA-CXCR7+SDF-1 groups.Real-time PCR was used to detect the mRNA expressions of TNF-α,IL-1β,IL-6 and IL-8.ELISA was used to detect the protein levels of TNF-α,IL-1β,IL-6 and IL-8 in the culture supernatant.Western blot was used to detect the protein expressions of NF-κB pathway.Results ① Transfection of SGC-7901 cells with CXCR7-shRNA lentiviral vector resulted in a significantly decreased expression of CXCR7 at both mRNA and protein levels (all P<0.01).② Compared with those in LV-shRNA-NC group,IL-6 and IL-8 mRNA expressions and protein levels in the culture supernatant were increased in LV-shRNA-NC+SDF-1 group (P<0.01 )and decreased in LV-shRNA-CXCR7 group (P<0.05).Compared with those in LV-shRNA-NC+SDF-1 group,the expressions of IL-6 and IL-8 at mRNA and protein levels in the culture supernatant were significantly cut down in LV-shRNA-CXCR7+SDF-1 group (P<0.01 ).However,the expressions of TNF-αand IL-1βat mRNA and protein levels in the culture supernatant were not significantly changed by SDF-1 and CXCR7 shRNA.③ The protein expressions of nuclear NF-κB p65,t-IκBαand p-IκBαexhibited no significant differences among the four groups.Conclusion SDF-1/CXCR7 can promote the synthesis and secretion of inflammatory cytokines IL-6 and IL-8 in gastric cancer SGC-7 9 0 1 cells through an NF-κB-independent pathway.