The Effects of miR-136-5p-Mediated Regulation of A20 in Astrocytes from Cultured Spinal Cord Cultured Cells In Vitro

Background/Aims: This study focused on investigating the regulatory mechanism of miR-136-5p in mouse astrocytes stimulated with interleukin-17(IL-17). Methods: C57BL/6 mouse astrocytes were stimulated with IL-17 (100ng/ml) for various periods of time (0-48 hours) and at various doses (0-200 ng), and the expression levels of inflammatory cytokine and chemokine genes (IL-6, TNF-α, MCP-1, MCP-5 and MIP-2) were then detected by real-time PCR. The expression of the A20 gene was measured with real-time PCR in cells that were stimulated with IL-17 (50 ng/ml) for various periods of time (0-48 hours). C57BL/6 mouse astrocytes were transfected with Ctrl-anti-miR-136-5p or LNA -anti-miR-136-5p for 48 h. Thereafter, the cells were stimulated with or without IL-17 (50ng/ml) for 6 h. The level of A20 protein (TNFα-induced protein 3, TNFAIP3) was detected by Western blot analysis. Results: (1) Compared with the DMEM control group, within six hours, IL-17 stimulation significantly increased the expression levels of inflammatory cytokine and chemokine genes and clearly decreased the expression level of the A20 protein. (2) Without IL-17 stimulation, the expression level of the miR-136-5p gene was significantly decreased, whereas in the miR-136-5p-inhibition group, the A20 protein expression was elevated. IL-17 stimulation slightly decreased the expression of the A20 protein in the miR-136-5p-inhibition group, but it was still slightly higher than in the control group. Conclusion: This study demonstrated that miR-136-5p affected the expression of A20 in IL-17-stimulated astrocytes.

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Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study

Reproductive Biology and Endocrinology ◽

10.1186/s12958-020-00692-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Song Guo ◽

Di Zhang ◽

Xiaowei Lu ◽

Qian Zhang ◽

Ruihuan Gu ◽

...

Keyword(s):

Real Time ◽

Western Blotting ◽

Real Time Pcr ◽

Underlying Mechanism ◽

Endometrial Receptivity ◽

Control Group ◽

Expression Levels ◽

Preliminary Study

Abstract Background Adenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM. Methods The study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting. Results Endometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice. Conclusions HIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM. Trial registration This trial was retrospectively registered.

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232 EFFECT OF SUPEROVULATION PRETREATMENT ON DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-FERTILIZED BOVINE EMBRYOS TRANSFERRED TO THE OVIDUCT-UTERUS ENVIRONMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab232 ◽

2016 ◽

Vol 28 (2) ◽

pp. 247

Author(s):

V. Havlicek ◽

A. Gad ◽

S. Papp ◽

K. Stein ◽

F. Palm ◽

...

Keyword(s):

Real Time ◽

Embryo Development ◽

Internal Control ◽

Real Time Pcr ◽

Control Group ◽

Hormonal Changes ◽

Expression Levels ◽

In Vivo Culture

Superovulation is a routine procedure to stimulate growth and ovulation of multiple follicles. However, the hormonal changes in the reproductive tract after superovulation treatment affect the environment and subsequently the early embryo development. The aim of the study was to examine the effect of superovulation pretreatment on embryo development and gene expression of IVM/IVF derived embryos subsequently cultured in vivo. The cumulus‐oocyte complexes derived from slaughterhouse ovaries were in vitro matured and fertilized. The denuded presumptive zygotes were cultured in CR1 medium with 5% oestrous cow serum. A total of 788 cleaved embryos at Day 2 were transferred by transvaginal endoscopy into the oviduct of synchronized and superovulated heifers (superstimulated group, SS) and 784 cleaved embryos were transferred into the ipsilateral oviduct of single ovulated synchronized heifers (single ovulation group, SO). In total, 10 Simmental heifers were used for in vivo culture in a crossover design. The in vivo culture was repeated once at an interval of at least 6 weeks in the same animal. At Day 7, embryos were recovered by combined flushing of the oviducts by endoscopy and the adjacent part of the uterine horns by conventional procedure. The numbers of recovered blastocysts were recorded and the embryos were cultured for the following 48 h to determine the blastocyst rate at Days 8 and 9. Simultaneously, 410 cleaved embryos were cultured in vitro for 9 days (control group, C). Triplicate pools of 10 blastocysts recovered at Day 7 from each treatment group were used for RNA isolation. Real-time PCR using sequence specific primers was performed in StepOnePlus™ real time PCR system (Applied Biosystem, Foster City, CA, USA). A comparative threshold cycle method was used to quantify expression levels of the candidate genes compared to the internal control GAPDH gene. The number of recovered embryos after in vivo culture was significantly lower in the SS group compared with the SO group (66.9 v. 79.5%, respectively; P < 0.05). The blastocyst rates at Days 7, 8, and 9 in the SS, SO, and C groups were not significantly different (31.9, 43.3, and 47.1% v. 35.2, 48.5, and 53.5% v. 37.8, 50, and 56.1%, respectively). Molecular analysis of selected genes playing important roles during pre-implantation development revealed significantly lower expression levels of IL6, IL18, and ABCC2 between both experimental in vivo culture groups and the C-group. The IL18 was also significantly down-regulated in the SS-group compared to the SO-group. The transcription factor NFκB was found to be down-regulated in the SS-group compared to the SO and C groups (P < 0.05). In conclusion, we showed that the superovulation pretreatment did not affect blastocyst yield during the culture period but seemed to influence the expression of developmentally important genes in the resulting embryos.

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Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2014-0004 ◽

2014 ◽

Vol 58 (1) ◽

pp. 23-28 ◽

Cited By ~ 3

Author(s):

Krzysztof Wąsowicz ◽

Piotr Podlasz ◽

Małgorzata Chmielewska ◽

Katarzyna Łosiewicz ◽

Jerzy Kaleczyc ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Experimental Colitis ◽

Expression Level ◽

Colonic Wall ◽

Brachyspira Hyodysenteriae ◽

Expression Levels ◽

Gapdh Expression ◽

Galanin Receptors ◽

Different Levels

Abstract The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

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4 MYOSTATIN GENE KNOCKDOWN THROUGH LENTIVIRAL VECTOR-MEDIATED DELIVERY OF shRNA FOR IN VITRO PRODUCTION OF TRANSGENIC BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab4 ◽

2007 ◽

Vol 19 (1) ◽

pp. 120 ◽

Cited By ~ 1

Author(s):

M. P. Milazzotto ◽

W. B. Feitosa ◽

B. E. Strauss ◽

M. Bajgelman ◽

C. M. Mendes ◽

...

Keyword(s):

Real Time ◽

Cell Morphology ◽

Real Time Pcr ◽

Lentiviral Vector ◽

Gene Knockdown ◽

Control Group ◽

Bovine Embryos ◽

Myostatin Gene ◽

Transgenic Embryo

The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed. This work was supported financially by FAPESP 03/0156-9.

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EGFL6 Modulates WNT Signaling to Drive Esophageal Cancer Metastasis and Proliferation

10.21203/rs.3.rs-30021/v1 ◽

2020 ◽

Author(s):

Yanhong Wang ◽

Jing Kang ◽

Jihua Tian ◽

Hongyan Jia ◽

Juanjuan Wang ◽

...

Keyword(s):

Esophageal Cancer ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Expression Level ◽

Marker Genes ◽

Invasion And Migration ◽

Ec Cells

Abstract Background Esophageal cancer (EC) is the sixth deadliest cancer in the world. There has been no breakthrough in the research on EC in the past few decades. Epidermal growth factor-like protein 6 (EGFL6), as a member of the epidermal growth factor superfamily, plays an important role in the occurrence and development of some tumors. However, the role of EGFL6 in the EC has never explored. Methods Immunohistochemical staining was used to evaluate the expression level of EGEC6 protein in human EC and its adjacent non-tumor tissues, and analyzed the correlation between the expression level of EGFL6 protein and clinical pathological indexes and survival rate. In vitro, by constructing EGFL6 silence and overexpressed EC cells,used CCK-8, clone formation, wound healing assays, transwell experiment and flow cytometry to explore the effects of EGFL6 on the proliferation, invasion, migration and apoptosis of EC. By using real-time PCR or western blot to detect the related marker genes of epithelial-mesenchymal transformation (EMT), tumor stem cells (TSCs) and Wnt/β-catenin. In vivo, established a nude mouse EC transplantation tumor model. Results The results showed that the expression level of EGFL6 in EC is significantly higher than that in adjacent non-tumor tissues, and is related to poor prognosis of patients. In vitro, CCK-8, clone formation, wound healing assays, transwell experiment and flow cytometry results show that EGFL6 overexpression can promotes proliferation, invasion and migration of EC cells and inhibits apoptosis. EGFL6 silencing inhibits proliferation, invasion and migration of EC cells and promotes apoptosis. Real-time PCR and Western-blot detection of EMT-related markers found that EGFL6 can induce EC cells EMT. Real-time PCR detection of esophageal cancer stem cell-related genes showed that EGFL6 may maintain the expression of esophageal cancer stem cell-like cell population. Western-blot detection of Wnt/β-catenin signaling marker genes showed that EGFL6 participated in the expression of Wnt/β-catenin signaling pathway. In vivo experiments found that knockout of EGFL6 could inhibit the formation of subcutaneous tumors in nude mice. Conclusion Taken together, our study identified a novel role and mechanism of EGFL6 in EC and provided epigenetic therapeutic strategies for the treatment of EC.

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An Efficient In Vitro Culture System To Amplify Spermatogonia Stem Cell Markers

Research in Molecular Medicine ◽

10.32598/rmm.8.3.2 ◽

2020 ◽

Vol 8 (3) ◽

pp. 117-124

Author(s):

Zeinab Narimanpour ◽

◽

Maryam Nazm Bojnordi ◽

Hatef Ghasemi ◽

◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Real Time ◽

Cultured Cells ◽

Expression Level ◽

Stem Cell Markers ◽

Testicular Cells ◽

Spermatogonia Stem Cell ◽

Alteration Pattern

Introduction: Proliferation of spermatogonial stem cells (SSCs) can be a treatment for infertile men. Here, we design an efficient method based on culturing in the presence of Sertoli cells to improve the expression level of some specific spermatogonia stem cell genes during two weeks post culture. Materials and Methods: Cells were derived from neonatal (2-6 days old) mice testes and were cultured in DMEM medium with FBS. The colonization of cultured SSCs in days 4, 7, and 14 of culture was counted via phase-contrast microscope and Image J software. Methyl thiazolyl tetrazolium (MTT) test was performed to evaluate the viability of cultured SSCs in days 3, 7, and 14 of culture. The expression level and the alteration pattern of specific spermatogonial markers, i.e., Stra8, DAZL, and Piwill2 was examined via real-time polymerase chain reaction (PCR) during two weeks post culture. Results: The number and the diameters of colonies showed a significant increase in cultured cells. MTT results proved the higher viability of testicular cells during the culture period. The results of ALP staining detected a positive reaction in spermatogonia colonies. Real-time PCR data showed that culturing SSCs in the presence of interstitial cells of the testis, amplified the level and alteration pattern of specific spermatogonia stem cells genes beneficial in the enrichment of SSCs propagation. Conclusion: Providing a similar culture environment to testicular niche increases viability, forms SSCs colonies, and regulates the level and alteration pattern of spermatogonia stem cell genes.

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The effect of BKV reactivation on cytokines behavior in kidney transplanted patients

BMC Nephrology ◽

10.1186/s12882-021-02645-y ◽

2022 ◽

Vol 23 (1) ◽

Author(s):

Zahra Rahimi ◽

Ramin Yaghobi ◽

Afsoon Afshari ◽

Jamshid Roozbeh ◽

Mohammad Javad Mokhtari ◽

...

Keyword(s):

Real Time ◽

Roc Curve ◽

Real Time Pcr ◽

Curve Analysis ◽

Expression Level ◽

Control Group ◽

Roc Curve Analysis ◽

Tnf Α ◽

Ifn Γ ◽

Inactive Group

Abstract Background BK virus associated nephropathy (BKVAN) is one of the common causes of graft loss among kidney transplanted recipients (KTRs). The current treatment for BKV nephropathy is decreasing the immunosuppressive regimen in KTRs. Interleukin-27 (IL-27) is a multifunctional cytokine that might be the front-runner of an important pathway in this regard. Therefore, in current study it is tried to evaluate the changes in the expression level of IL-27 and some related molecules, resulting from BKV reactivation in KTR patients. Methods EDTA-treated blood samples were collected from all participants. Patients were divided into two groups, 31 kidney transplant recipients with active and 32 inactive BKV infection, after being monitored by Real time PCR (Taq-Man) in plasma. Total of 30 normal individuals were considered as healthy control group. Real time PCR (SYBR Green) technique is used to determine the expression level of studied genes. Results The results of gene expression comparisons showed that the expression level of IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 genes was significantly higher in inactive group in comparison to active group. The expression level of TLR4 was lower in both active and inactive groups in comparison to control group. ROC curve analysis showed that IL-27 and IRF7 are significantly different amongst other studied genes. Finally, the analyses revealed that the expression level of most of the studied genes (except for TNF-α and TLR4) have significant correlation with viral load. Conclusions Our findings revealed that IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 expression level is higher in inactive group and TLR4 expression level is lower in patients’ groups in comparison to control group. Also, ROC curve analysis showed IL-27 and IRF7 can significantly differentiate studied groups (BKV active vs. inactive). Therefore, these results might help elucidating the pattern in charge of BKV reactivation in kidney transplanted patients.

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Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes

Archives of Iranian Medicine ◽

10.34172/aim.2021.86 ◽

2021 ◽

Vol 24 (8) ◽

pp. 607-614

Author(s):

Maryam Samareh Salavati Pour ◽

Fatemeh Hoseinpour Kasgari ◽

Alireza Farsinejad ◽

Ahmad Fatemi ◽

Gholamhossein Hassanshahi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Real Time ◽

Real Time Pcr ◽

Treated Group ◽

Population Doubling ◽

Control Group ◽

Population Doubling Time ◽

Longevity Genes

Background: Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Methods: Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Results: Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. Conclusion: According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

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High FLT3 mRNA Expression in Acute Myeloid Leukemia May Be Functionally an Alternative to Mutational Activation of the Receptor.

Blood ◽

10.1182/blood.v104.11.3374.3374 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3374-3374 ◽

Cited By ~ 1

Author(s):

Florian C. Kuchenbauer ◽

Wolfgang Kern ◽

Claudia Schoch ◽

Alexander Kohlmann ◽

Wolfgang Hiddemann ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

De Novo ◽

Expression Level ◽

Alternative Mechanism ◽

Expression Levels ◽

Flt3 Mutation ◽

Flt3 Mutations ◽

Flt3 Expression ◽

Mutational Activation

Abstract Mutations of the FLT3 gene are detectable in approximately 30% of all adult AML. These mutations lead to an autoactivation of the receptor inducing increased proliferation of the leukemic clone. An alternative mechanism of FLT3 activation might be mRNA overexpression. In the presented study the FLT3 expression level in 208 adult AML patients and 8 healthy donors was assessed by real time PCR and correlated to several parameters. In all patients cytomorphology, cytogenetics, and FLT3 mutation status was assessed. Significant differences of FLT3-expression levels were found in certain AML subgroups. The highest expression levels were found in FAB subtypes M5 and the lowest in M3. In total, increasing levels were shown in the following order: M3 <M3v <M6 <M2 <M4eo <M4 <M0 <M1 <M5a <M5b. Independent analysis of FLT3 expression in different cytogenetic AML subgroups showed the lowest median in the t(15;17) group, followed by t(8;21), inv(16), normal, complex karyotype and the highest median in the t(11q23) group. No difference was observed between the group of secondary AML following MDS, therapy related AML and the de novo AML (p=0.868, p=0.562, and p=0.570, respectively). Compared to clinical parameters, FLT3 expression correlated with high percentage of bone marrow blasts (p=0.0005) and high leukocyte count (p<0.001). In contrast to previous studies no difference in FLT3 expression levels was detected between AML with (n=74) and without (n=130) any FLT3 mutation. Assessment of FLT3 RNA by microarray analysis and FLT3 receptor surface expression (CD135) detected by flow cytometry correlated significantly with FLT3-expression as assessed by real time PCR (p<0.001, each). To analyze whether high FLT3 expression is a prognostic parameter 118 AML cases with normal karyotype were devided into two groups. Group 1 (n=75) was defined to have less and group 2 (n=43) more than the median of the FLT3 expression level found in the total group. No impact on OS and EFS could be shown (608 vs. 311 days, p=0.1283 and 398 vs. 208 day, p=0.3056). In conclusion, these data support the hypothesis that FLT3 activation through mRNA overexpression is an alternative mechanism to FLT3 mutations. Especially as it was found extremely high in 11q23 AML, that rarely reveal FLT3 mutations.

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Fuyuan Xingnao Decoction Promotes Angiogenesis Through the Rab1/AT1R Pathway in Diabetes Mellitus Complicated With Cerebral Infarction

Frontiers in Pharmacology ◽

10.3389/fphar.2021.616165 ◽

2021 ◽

Vol 12 ◽

Author(s):

Dong Deng ◽

Yao Qu ◽

Lihua Sun ◽

Liyang Jia ◽

Jianhong Bu ◽

...

Keyword(s):

Diabetes Mellitus ◽

Cerebral Cortex ◽

Cerebral Infarction ◽

Real Time ◽

Control Group ◽

Ang Ii ◽

Cerebral Microvessels ◽

Expression Levels

Fuyuan Xingnao decoction (FYXN), a traditional Chinese formula comprised of seven herbs, has been utilized to treat diabetes mellitus complicated with cerebral infarction (DMCI) for years. Yet, its protective and regulatory mechanism is poorly understood. The aim of the study is to investigate the effects of FYXN on DMCI in vitro and in vivo, as well as its mechanism in angiogenesis. For in vivo experiments, FYXN was administered to DMCI rats with streptozotocin (STZ) injection-induced diabetes. Then middle cerebral artery occlusion (MCAO) was conducted and the cerebral cortex sections of the rats were obtained. The ultrastructure of cerebral microvessels and new vessel density of ischemic penumbra were evaluated by the transmission electron microscopy (TEM) assay and immunohistochemistry, respectively. Protein and mRNA expression levels of Rab1/AT1R in cortex were assayed by Western blotting and real-time fluorescence quantitative real-time polymerase chain reaction (RT-qPCR). In vitro, FYXN serum was produced in rats on the fourth day 2 h after the last FYXN administration. Green fluorescence was observed after transfection with lentivirus packaged Rab1-WT or siRNA for 24 h. The activity of brain microvascular endothelial cells (BMECs) treated with sera from these rats was tested by MTT assay and Transwell assays, respectively. The expression of AT1R on the cell membrane and endoplasmic reticulum of BMECs was evaluated by immunofluorescence staining. Protein expression levels of signaling molecules in the Rab1/AT1R pathways were also detected. Results showed that in vivo, FYXN treatment significantly intensified CD31 staining in the cortical areas and enhanced the mRNA and protein levels of AT1R, Ang II, Rab1a, Rab1b and VEGF expression in ischemic cerebral cortex tissues. In vitro, the expression levels of AT1R, Ang II, Rab1a, Rab1b and VEGF in the cerebral infarction model group were significantly higher than those in the control group, with further increases after administration of FYXN drug serum. FYXN promoted the proliferation and migration of BMECs by activating the Rab1/AT1R signaling pathway. In conclusion, FYXN exerts a protective effect against DMCI by promoting angiogenesis via the Rab1/AT1R pathway, which provides strong evidence for the therapeutic effect of FYXN on DMCI.

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