scholarly journals miR-122-5p regulates the tight junction of the blood-testis barrier of mice via occludin

2021 ◽  
Vol 31 (1) ◽  
Author(s):  
Limin Liu ◽  
Maoying Zhu ◽  
Xiaoli Liu ◽  
Lumin Fei ◽  
Jianyun Shen ◽  
...  

Abstract Background Occludin protein is the primary assembling protein of TJs and the structural basis for tight junction formation between Sertoli cells in the spermatogenic epithelium. The expression of miR-122-5p and occludin are negatively correlated. In order to investigate the regulation mechanism of miR-122-5p on occludin and TJ, the present study isolated primary Sertoli cells from C57BL/6 mice, identified a transcription factor of miR-122-5p in testicle, studied the modulating loci of miR-122-5p on occludin using a dual-luciferase reporter assay, analyzed the regulate of miR-122-5p on the expression of occludin with real-time RT-PCR and Western blot, and studied the effect of miR-122-5p on the tight junction using a Millicell Electrical Resistance System. Results The relative luciferase activity in the pcDNA-Sp1 + pGL3-miR-122-5p promoter group was significantly higher than that in the pcDNA-Sp1 + pGL3-basic group, which suggests that transcript factor Sp1 promotes the transcription of miR-122-5p. The relative luciferase activity in the occludin 3′-UTR (wt) + miR-122-5p mimic group was significantly lower than that in the other groups (p < 0.01), which indicates that miR-122-5p modulates the expression of occludin via the ACACTCCA sequence of the occludin-3’UTR. The levels of occludin mRNA and protein in the miR-122-5p mimic group were significantly lower than that in the other groups (p < 0.05), which indicates that miR-122-5p reduces the expression of occludin. The trans-epithelial resistance of the miR-122-5p mimic group was significantly lower than that of the blank control group after day 4 (p < 0.05), which indicates that miR-122-5p inhibited the assembly of the inter-Sertoli TJ permeability barrier in vitro. Conclusion These results displayed that miR-122-5p could regulate tight junctions via the Sp1-miR-122-5p-occludin-TJ axis.

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10374
Author(s):  
Ying Jin ◽  
Xiaoyan Sun ◽  
Fang Pei ◽  
Zhihe Zhao ◽  
Jeremy Mao

Background Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. Methods We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. Results Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. Conclusion Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.


2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Jing Gao ◽  
Jie Yin ◽  
Kang Xu ◽  
Hui Han ◽  
ZeMin Liu ◽  
...  

Infantile diarrhea is a serious public health problem around worldwide and results in millions of deaths each year. The levels and sources of dietary protein are potential sources of diarrhea, but the relationship between the pathogenesis causes of infantile diarrhea and protein intake remains poorly understood. Many studies have indicated that the key to understanding the relationship between the protein in the diet and the postweaning diarrhea of piglets is to explore the influences of protein sources and levels on the mammalian digestion system. The current study was designed to control diarrhea control by choosing different protein levels in the diet and aimed at providing efficient regulatory measures for infantile diarrhea by controlling the protein levels in diets using a postweaning piglets model. To avoid influences from other protein sources, casein was used as the only protein source in this study. Fourteen piglets (7.98±0.14 kg, weaned at 28 d) were randomly allotted to two dietary treatments: a control group (Cont, containing 17% casein) and a high protein group (HP, containing 30% casein). The experiment lasted for two weeks and all animals were free to eat and drink water ad libitum. The diarrhea score (1=normal; 3=watery diarrhea) and growth performance were recorded daily. The results showed that the piglets in HP group had persistent diarrhea during the whole study, while no diarrhea was noticed in the control groups. Also, the feed intake and body weights were reduced in the HP groups compared with the other group (P<0.05). The diarrhea-related mRNA abundances were analyzed by real-time PCR; the results showed that HP treatment markedly decreased the expression of aquaporin (AQP, P<0.05) and the tight junction protein (P<0.05), but increased inflammatory cytokines (P<0.01) than those in control group. In addition, the Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway (P<0.01) was inhibited in the HP group. Intestinal microbiota was tested by 16S sequencing, and we found that the HP group had a low diversity compared the other group. In conclusion, despite being highly digestible, a high casein diet induced postweaning diarrhea and reduced the growth performance of the postweaning piglets. Meanwhile, AQP, tight junction protein, and intestinal immune were compromised. Thus, the mechanism of how a highly digestible protein diet induces diarrhea might be associated with the AMPK signaling pathway and intestinal microbiome.


Endocrinology ◽  
2001 ◽  
Vol 142 (5) ◽  
pp. 1878-1888 ◽  
Author(s):  
Nancy P. Y. Chung ◽  
C. Yan Cheng

Abstract The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl2-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 × 106 cells/cm2) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl2 at 5–10μ m for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl2 (0.1–5 μm) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl2 also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl2, the presence of testosterone (T) at 1 × 10−9m allowed resealing of the inter-Sertoli TJ barrier after CdCl2 was removed, whereas the presence of 2 × 10−7m testosterone even protected Sertoli cells from CdCl2-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl2-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl2 treatment. Based on these results, it is apparent that CdCl2-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.


2013 ◽  
Vol 50 (3) ◽  
pp. 305-318 ◽  
Author(s):  
Wenhui Su ◽  
Xinchun Liu

In mammalian testes, the blood–testis barrier (BTB), created by specialized junctions between Sertoli cells near the basement membrane of the seminiferous epithelium, provides an indispensable immune-privileged microenvironment for spermatid development. However, the BTB must experience restructuring during the epithelial cycle to facilitate the transit of preleptotene spermatocytes upon the testosterone-induced new TJ fibrils forming behind these cells, which is intimately related to the extensive dynamics of junction protein complexes between Sertoli cells. As key regulators of protein traffic, Rab GTPases participate in delivery of proteins between distinct cellular sites and cross talk with proteins that constitute tight junction and adherens junction. Using primarily cultured Sertoli cellsin vitrowith an established tight junction permeability barrier that mimics the BTBin vivo, RAB13 was shown to decrease during the testosterone-induced TJ integrity enhancement, accompanied with an increment in protein kinase A (PKA) activity. Furthermore, knockdown ofRab13was found to resemble the effect of testosterone on Sertoli cell TJ permeability by reinforcing filamentous actin and occludin distribution at the cell–cell interface and promoting the direct interaction between ZO-1 and occludin. Interestingly, the effects of testosterone andRab13knockdown on Sertoli cell epithelium were revealed to be antagonized by PKA activity inhibition. In summary, RAB13 serves as a regulatory component in the assembly and restructuring of the TJ fibrils between adjacent Sertoli cells.


Author(s):  
A.J. Verkleij

Freeze-fracturing splits membranes into two helves, thus allowing an examination of the membrane interior. The 5-10 rm particles visible on both monolayers are widely assumed to be proteinaceous in nature. Most membranes do not reveal impressions complementary to particles on the opposite fracture face, if the membranes are fractured under conditions without etching. Even if it is considered that shadowing, contamination or fracturing itself might obscure complementary pits', there is no satisfactory explanation why under similar physical circimstances matching halves of other membranes can be visualized. A prominent example of uncomplementarity is found in the erythrocyte manbrane. It is wall established that band 3 protein and possibly glycophorin represents these nonccmplanentary particles. On the other hand a number of membrane types show pits opposite the particles. Scme well known examples are the ";gap junction',"; tight junction, the luminal membrane of the bladder epithelial cells and the outer membrane of Escherichia coli.


2017 ◽  
Vol 68 (2) ◽  
pp. 323-327
Author(s):  
Cristian Levente Giuroiu ◽  
Maria Vataman ◽  
Gabriel Melian ◽  
Dragos Bularda ◽  
Ludmila Lozneanu ◽  
...  

The study aimed to assess the number, localization and distribution of interleukin 6 (IL-6) positive cells in healthy pulp, acute and chronic pulpitis. The study group included 48 patients aged between 18-72, treated in University of Medicine and Pharmacy Grigore T. Popa Iasi, Romania. The pulpectomy was performed on 42 patients diagnosed with acute and chronic pulpitis. The other 6 patients, without signs of dental caries or periodontal disease, were submitted to extractions of teeth for orthodontic purposes, with pulpectomy performed before extraction. The pulp samples were examined with optic microscope. The detection and assessment of IL-6 were performed using immunohistochemical technique. Data were statistically analysed using non-parametric tests. According to morphopathological criteria, 42.85% were classified as acute pulpitis and 57.14% as chronic pulpitis. The pulp samples in control group were not associated with IL-6 positive cells. The analysis of all samples with acute and chronic pulpitis identified 73.80% samples with IL-6 and 26.20% associated with the absence of IL-6. The highest frequency of IL-6 positive cells was recorded in rich-cell zone of crown dental pulp. The systemic distribution of IL-6 positive cells was mostly diffused without well-defined orientation. IL-6 release in acute and chronic pulpitis is significantly higher comparing with healthy pulp tissue.


2018 ◽  
Vol 69 (8) ◽  
pp. 2081-2088 ◽  
Author(s):  
Alin Alexandru Odor ◽  
Edwin Sever Bechir ◽  
Deborah Violant ◽  
Victoria Badea

Moderate and severe periodontitis represents a challenge in the non-surgical periodontal therapy. Due to the lack of evidence regarding the antimicrobial effectiveness of 940 nm diode laser in periodontal treatment, this study aimed to evaluate the antimicrobial effect of hydrogen peroxide (H2O2) photolysis performed with 940 nm diode laser in the treatment of moderate and severe periodontitis. Twenty-five patients with 100 teeth were selected for this pilot study. The test teeth were randomly assigned to one of the four treatment groups: Group 1: scaling and root planning (SRP) (control group); and the following experimental groups: Group 2: H2O2; Group 3: 940 nm diode laser therapy; Group 4: 940 nm diode laser therapy and H2O2. Clinical examinations, like probing depth (PD), clinical attachment level (CAL) and bleeding on probing (BOP) were performed before and after the treatment. The microbiological evaluation, effectuated before and after the treatment, included nine periodontal bacteria species and investigated by means of real-time PCR assay. The clinical and bacterial differences in the tested groups, was assessed between control group and the other three experimental groups, as well as between the experimental groups. The total bacteria load was reduced for all four studied groups. Group 4 (diode laser + H2O2) showed significant bacterial reduction of the major periodontal bacteria like Pg., Tf., Td., Pi., Pm., Fn (p[0.001) than the other 3 groups (p]0.001). Also the periodontal clinical parameters, like PD, CAL and BOP showed a significant reduction after the photolysis of H2O2 with the 940 nm diode laser (p[0.001). Differences between tested groups showed a significant beneficial results in regard to Group 4.It is suggested that the photoactivation of H2O2 with the 940 nm diode laser can be used successfully in adjunctive to the non-surgical periodontal treatment as a bactericidal tool.


Author(s):  
Zheng Dong ◽  
Qing-Hua Xu ◽  
Yuan-Bin Zhu ◽  
Yong-Feng Wang ◽  
Jie Xiong ◽  
...  

Aims : The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). Methods: A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment was selected. The expression of miR-22 was detected by fluorescence quantitative real-time PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGF-D) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. Results: The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 cotransfection group reversed the changes. Conclusion: We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.


Author(s):  
Jiyune Yi ◽  
Seul Gee Kim ◽  
Taegyu Khil ◽  
Minja Shin ◽  
Jin-Hee You ◽  
...  

We developed two distinct forest therapy programs (FTPs) and compared their effects on dementia prevention and related health problems for older adults. One was focused on Qigong practice in the forest (QP) and the other involved active walking in the forest (WP). Both FTPs consisted of twelve 2-h sessions over six weeks and were conducted in an urban forest. We obtained data from 25, 18, and 26 participants aged 65 years or above for the QP, WP, and control groups, respectively. Neuropsychological scores via cognition (MoCA), geriatric depression (GDS) and quality of life (EQ-5D), and electrophysiological variables (electroencephalography, bioimpedance, and heart rate variability) were measured. We analyzed the intervention effects with a generalized linear model. Compared to the control group, the WP group showed benefits in terms of neurocognition (increases in the MoCA score, and alpha and beta band power values in the electroencephalogram), sympathetic nervous activity, and bioimpedance in the lower body. On the other hand, the QP group showed alleviated depression and an increased bioimpedance phase angle in the upper body. In conclusion, both active walking and Qigong in the forest were shown to have distinctive neuropsychological and electrophysiological benefits, and both had beneficial effects in terms of preventing dementia and relieving related health problems for elderly individuals.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zheng Zheng ◽  
Yan Chen ◽  
Yinzhou Wang ◽  
Yongkun Li ◽  
Qiong Cheng

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.


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