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To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.
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Animals , Mice , Mice, Knockout , CRISPR-Cas Systems , Blood Pressure , Gene Knockout Techniques , ZygoteABSTRACT
OBJECTIVE@#To observe the effect of acupoint thread-embedding on tight junction of intestinal mucosal epithelial barrier in rats with ulcerative colitis (UC) under the state of "deficiency and stasis", and to explore its mechanism.@*METHODS@#Sixty male SD rats were randomly divided into a control group (@*RESULTS@#Compared with the control group, in the model group the body weight was decreased (@*CONCLUSION@#The thread-embedding could repair the tight junction of intestinal mucosa epithelium and reduce the permeability of intestinal mucosa epithelium, which may be related to the decrease of the expression of CaMKⅡ, MLCK and other protein kinases.
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Animals , Male , Rats , Acupuncture Points , Colitis, Ulcerative/therapy , Epithelium , Intestinal Mucosa , Rats, Sprague-Dawley , Tight JunctionsABSTRACT
Objective: To explore the effect of herb-partitioned moxibustion (HPM) on tight junctions (TJs) of intestinal epithelial cells in Crohn disease (CD) mediated by tumor necrosis factor-α (TNF-α)-nuclear factor kappa B (NF-κB)-myosin-light- chain kinase (MLCK) pathway. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an HPM group and a mesalazine (MESA) group, with 12 rats in each group. Trinitrobenzene sulfonic acid (TNBS) was administered to establish CD models. When the model was confirmed a success, the HPM group rats were treated with HPM at Tianshu (ST 25) and Qihai (CV 6), while the MESA group rats were given MESA solution by lavage. When the intervention finished, the colonic epithelial tissues were separated, purified and cultured in each group to establish the intestinal epithelial barrier model in vitro, and TNF-α was added (100 ng/mL) in the culture medium and maintained for 24 h to establish an increased epithelial permeability model. Transepithelial electrical resistance (TEER) was used to examine the permeability of the barrier; Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway; immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium. Results: After TNF-α induction, compared with the MC+TNF-α group, the TEER value increased significantly in the HPM+TNF-α and MESA+TNF-α groups (both P<0.001); the expressions of nuclear factor kappa B (NF-κB) p65, MLCK, myosin light chain (MLC), tumor necrosis factor receptor-associated factor 6 (TRAF6) and receptor interaction protein-1 (RIP1) decreased significantly (P<0.01 or P<0.05), and the expression of zinc finger protein A20 (A20) increased significantly (P<0.01); the expressions of occludin, claudin-1, zonula occludens protein 1 (ZO-1) and F-actin also increased significantly (all P<0.01). Compared with the MESA+TNF-α group, the expressions of MLC, occludin, claudin-1, ZO-1 and F-actin increased significantly in the HPM+TNF-α group (P<0.01 or P<0.05). Conclusion: HPM can protect or repair the damage of intestinal epithelial barrier in CD rats, which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.
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Myosin light chain 9 (MYL9) is a regulatory light chain of myosin, which plays an important role in various biological processes including cell contraction, proliferation and invasion. MYL9 expresses abnormally in several malignancies including lung cancer, breast cancer, prostate cancer, malignant melanoma and others, which is closely related to the poor prognosis, but the clinical significance for its expression varies with different types of cancer tissues. Further elucidating the molecular mechanism of MYL9 in various types of malignant tumor metastasis is of great significance for cancer prevention and treatment. At the same time, as a molecular marker and potential target, MYL9 may have great clinical value in the early diagnosis, prognosis prediction, and targeted treatment of malignant tumors.
Subject(s)
Humans , Male , Biomarkers , Lung Neoplasms , Myosin Light Chains/metabolism , Prognosis , Prostatic NeoplasmsABSTRACT
Objective:To explore the possible mechanism of electroacupuncture to improve detrusor hyperreflex after suprasacral spinal cord injury. Methods:A total of 60 female Sprague-Dawley rats were included. According to the random number table, twelve were selected as the blank group, twelve as the sham operation group, and the remaining 36 were made neurogenic bladder models using modified T10 spinal cord transection. After that, twelve of them were randomly selected as the model group and twelve were as the electroacupuncture group from the model rats that met the requirements. On the 19th day after modelling, Ciliao (BL32), Zhongji (RN3) and Sanyinjiao (SP6) were taken for electroacupuncture. After seven days of continuous treatment, urodynamic testing was performed, content of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in detrusor was determined by ELISA, and the level of phosphorylation of myosin light chain kinase (p-MLCK) of detrusor was determined by Western blotting. Results:Compared with the blank group and the sham operation group, the maximum bladder capacity and bladder compliance significantly reduced (P < 0.01), and the base pressure and leakage point pressure of bladder significantly increased (P < 0.01); the content of cAMP and PKA in detrusor reduced (P < 0.01), p-MLCK in detrusor reduced (P < 0.05) in the model group. Compared with the model group, the maximum bladder capacity and bladder compliance increased (P < 0.01), the base pressure of the bladder and the pressure at the leak point decreased (P < 0.05); the contents of cAMP and PKA protein in detrusor increased (P < 0.05), the p-MLCK in detrusor increased (P < 0.05) in the electroacupuncture group. Conclusion:Electroacupuncture at Ciliao, Zhongji and Sanyinjiao points could improve the bladder function of rats with detrusor hyperreflex after complete spinal cord injury, and its mechanism may be related to up-regulating the expression of cAMP and PKA, phosphorylating and inactivating p-MLCK, which promote relaxation of detrusor.
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OBJECTIVE@#To investigate the effect of Chang'an II Decoction ( II ))-containing serum on intestinal epithelial barrier dysfunction in rats.@*METHODS@#Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium. Caco-2 monolayers were treated with blank serum and Chang'an II Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang'an II Decoction intragastrically at doses of 0.49, 0.98, 1.96 g/(kg·d) for 1 week, respectively. After preparation of containing serum, cells were divided into the normal group, the model group, the Chang'an II-H, M, and L groups (treated with 30 ng/mL TNF-α and medium plus 10% high, middle-, and low-doses Chang'an II serum, respectively). Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-labeled dextran. Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs). Immunofluorescence of zonula occludens-1 (ZO-1), claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution. The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction. The expression levels of MLCK, myosin light chain (MLC) and p-MLC were determined by Western blot.@*RESULTS@#Chang'an II Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α. It alleviated TNF-α-induced morphological alterations in TJ proteins. The increases in MLCK mRNA and MLCK, MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang'an II-H group. Additionally, Chang'an II Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus.@*CONCLUSION@#High-dose Chang'an II-containing serum attenuates TNF-α-induced intestinal barrier dysfunction. The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.
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Objective@#To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism.@*Methods@#Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and t test.@*Results@#After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (t=18.404, 9.140, P<0.01) and negative control group (t=15.099, 7.096, P<0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=21.140, 5.440, P<0.01) and negative control group (t= 14.310, 5.210, P<0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=19.160, 7.710, P<0.01) and negative control group (t= 19.890, 7.500, P<0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.709, 4.011, P<0.05 or P<0.01) and negative control group (t=2.373, 3.744, P<0.05 or P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=4.285, 5.050, P<0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=9.118, 11.313, P<0.01) and negative control group (t=9.073, 11.280, P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (t=2.889, 2.640, P<0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.652, 3.983, P<0.05 or P<0.01) and negative control group (t=2.792, 4.065, P<0.05 or P<0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=3.881, 3.570, P<0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (t=5.011, 3.982, P<0.05 or P<0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (t= 4.880, 3.862, P<0.05 or P<0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (t=4.178, 3.830, P<0.05 or P<0.01).@*Conclusions@#HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells.
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Objective:To investigate the effect of endothelin-1 (ET-1) on the expression of phosphorylated myosin light chain Ⅱ(p-MLCⅡ)and myosin light chain Ⅱ(MLCⅡ)protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San(DSS)drug-containing serum. Method:After HSC-T6 cells were seeded, DMEM and blank rat serum with final concentrations of 2.5%, 5%, 10%, 15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium(MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group (5%, 10%, 15%) and DSS drug-containing serum group (5%, 10%, 15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), DSS drug-containing serum low (5%), medium (10%) and high dose (15%) groups. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), model group (10%), DSS drug-containing serum low (5%), medium (10%), high dose (15%) groups and Y-27632 inhibitor group (100 μmol·L-1). Except the blank serum control group, the other groups all received 10 nmol·L-1 ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡ and MLCⅡ in HSC-T6 cells induced by ET-1. Result:Serum concentrations of 5%, 10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group, the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state (PPPPPConclusion:DSS drug-containing serum may down-regulate the expression of p-MLCⅡ and MLCⅡ by down-regulating the content of ET-1 and inhibiting the autocrine of ET-1.
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Coronary artery spasm(CAS)is a hyper-contraction of segmental coronary artery in response to multiple stimuli. At present, it's still in lack of specific diagnostic indicators of sudden cardiac death caused by CAS. This review summarizes current researches on the mechanisms of CAS and describes the roles of vascular endothelial dysfunction and vascular smooth muscle hypersensitivity in the course of CAS. Furthermore, the molecular mechanisms of the endogenous NO and endothelin-1 cause vascular endothelial dysfunction, and the phosphorylation of MLC2, Rho kinase and endoplasmic reticulum stress related to vascular smooth muscle hypersensitivity are discussed. Meanwhile, the possibility of forensic application for the related molecules on the diagnosis of sudden cardiac death caused by CAS are also explored.
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Objective:To explore the effect of differentiation of cardiac stem cells(CSC)mediated by pilose antler polypeptides on the expressions of terminal myocardial differentiation genes atrial natriuretic polypeptide (ANP)and myosin light chain 2v(MLC-2v),and to clarify the mechanism of repairing the damaged myocardium.Methods:The healthy male Wistar rats born 2 d were selected to extract the CSC.The surface marker c-Kit and the purity of CSC were identified by immunofluorescence and flow cytometry.The culture dish was used as the experimental unit and the extracted cells were divided into the following four groups:blank control group(the same amount of buffer was added for induction),5-azacytidine group(induced with 3 μmol·L-15-azacytidine),pilose antler polypeptides group(induced with 800 mg·L-1pilose antler polypeptides)and combined group(induced with 800 mg·L-1pilose antler polypeptides and 3 μmol·L-15-azacytidine);the cells were incubated for 48 h in the condition of 37℃ and 5% CO2.The expression levels of ANP and MLC-2v in supernatant of the cells in various groups were detected by ELISA method.The expression levels of ANP and MLC-2v mRNA in the cells in various groups were detected by RT-PCR method.Results:CSC were prepared with the purity>95%.The results of ELISA showed that the expression levels of ANP and MLC-2v in 5-azacytidine,pilose antler polypeptides and combined groups were significantly increased compared with blank control group(P<0.05).The expression levels of ANP and MLC-2v in combined group were increased compared 5-azacytidine and pilose antler polypeptides groups,but there were no significant differences(P>0.05).The result of RT-PCR showed that the expression levels of ANP and MLC-2v mRNA in 5-azacytidine,pilose antler polypeptides and combined groups were significantly increased compared with blank control group(P<0.05).Conclusion:Pilose antler polypeptides can induce the differentiation of CSC into cardiac cells by promoting the expressions of ANP and MLC-2v,and they can reduce the cytotoxicity induced by 5-azacytidine in some degrees.
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OBJECTIVE: To analyze the correlation between vascular endothelia injury factors and occupational hand-arm vibration disease( HAVD). METHODS: A judge sampling method was used to select 23 male patients with HAVD as the HAVD group,61 male workers who exposed to hand-arm vibration without HAVD as the vibration exposure group,64 male workers without hand-arm vibration exposure as the control group. The plasma levels of myosin light chain 2( MLC2),endothelin-1( ET-1) and vinculin( VCL) were detected by enzyme-linked immunosorbent assay. Logistic regression analysis was used to analyze the related indicators of HAVD for building the new multivariable model index Y. The indicators of HAVD were screened and judged by receiver operating characteristic( ROC) curves. RESULTS: There was significant difference in plasma levels of MLC2 among the three groups( P < 0. 05). The levels from high to low was as follows: HAVD group > vibration exposure group > control group. The plasma level of ET-1 in HAVD group was lower than that in the control group( P < 0. 05),but there was no significant difference between vibration exposure group and HAVD group( P > 0. 05). There was no significant difference among the three groups in the plasma level of VCL( P > 0. 05).The logistic regression analysis results showed that after adjusting confounding factors such as age,length of service,smoking,alcohol drinking and subjective symptoms,the higher MLC2 plasma level,the higher risk of HAVD( P < 0. 01),and the lower ET-1 plasma level,the higher risk of HAVD( P < 0. 05). According to ROC curve analysis,the area under the ROC curve( A_Z) value of the plasma levels of MLC2 and ET-1 were 0. 820 and 0. 524,respectively( P < 0. 01). The predictive probability index Y built with MLC2 and ET-1 by logistic regression model was used to judge the A_Z value of HAVD to be 0. 799( P < 0. 01). The A_Z values from high to low was as follows: MLC2 > Y> ET-1( P < 0. 01).CONCLUSION: The plasma levels of MLC2 and ET-1 are correlated with HAVD. The efficacy of MLC2 as a biomarker for screening HAVD is better than that of ET-1. No association was found between VCL and HAVD.
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Objective: To explore the effect of differentiation of cardiac stem cells (CSC) mediated by pilose antler polypeptides on the expressions of terminal myocardial differentiation genes atrial natriuretic polypeptide (ANP) and myosin light chain 2v (MLC-2v), and to clarify the mechanism of repairing the damaged myocardium Methods: The healthy male Wistar rats born 2 d were selected to extract the CSC. The surface marker c-Kit and the purity of CSC were identified by immunofluorescence and flow cytometry. The culture dish was used as the experimental unit and the extracted cells were divided into the following four groups: blank control group (the same amount of buffer was added for induction), 5-azacytidine group (induced with 3 jumol · L-1 5-azacytidine), pilose antler polypeptides group (induced with 800 mg · L-1 pilose antler polypeptides) and combined group (induced with 800 mg · L-1 pilose antler polypeptides and 3 μmol · L-1 5-azacytidine); the cells were incubated for 48 h in the condition of 37°C and 5% CO2. The expression levels of ANP and MLC-2v in supernatant of the cells in various groups were detected by ELISA method. The expression levels of ANP and MLC-2v mRNA in the cells in various groups were detected by RT-PCR method. Results: CSC were prepared with the purity0.05). The result of RT-PCR showed that the expression levels of ANP and MLC-2v mRNA in 5-azacytidine, pilose antler polypeptides and combined groups were significantly increased compared with blank control group (P<0.05). Conclusion: Pilose antler polypeptides can induce the differentiation of CSC into cardiac cells by promoting the expressions of ANP and MLC-2v, and they can reduce the cytotoxicity induced by 5-azacytidine in some degrees.
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α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms, but the mechanism responsible for this activity has not been determined. To determine the role played by ICB on the regulation of vascular tone, we investigated the inhibitory effects of ICB on vascular contractile responses by adrenergic α-receptor agonists. In addition, we investigated the role on myosin light chain (MLC) phosphorylation and cytosolic calcium concentration in vascular smooth muscle cells (VSMC). In aortic rings isolated from C57BL/6J mice, ICB significantly attenuated the contraction induced by phenylephrine (PE) and norepinephrine (NE), whereas ICB had no effects on KCl (60 mM)-induced contraction. In vasculatures precontracted with PE, ICB caused marked relaxation of aortic rings with or without endothelium, suggesting a direct effect on VSMC. In cultured rat VSMC, PE or NE increased MLC phosphorylation and increased cytosolic calcium levels. Both of these effects were significantly suppressed by ICB. In conclusion, our results showed that ICB regulated vascular tone by inhibiting MLC phosphorylation and calcium flux into VSMC, and suggest that ICB has anti-hypertensive properties and therapeutic potential for cardiovascular disorders related to vascular hypertension.
Subject(s)
Animals , Mice , Rats , Aorta, Thoracic , Calcium , Cytosol , Endothelium , Hypertension , Lignin , Muscle, Smooth, Vascular , Myosin Light Chains , Myosins , Norepinephrine , Phenylephrine , Phosphorylation , Plants, Medicinal , Relaxation , SchisandraABSTRACT
Aim To investigate the protective effect of paeoniflorin on TNF-α induced intestinal epithelial barrier dysfunction and its mechanisms.Methods The Caco-2 cells were cultured and the MTF assay was used to determine the effects of the paeoniflorin on Caco-2 cell activity.The Caco-2 cell intestinal epithelial barrier dysfunciton model was established through incubation of cells with TNF-α.The effects of paeoniflorin on intestinal epithelial barrier dysfunciton were studied.Results The transmembrane resistance in Caco-2 epithelial barrier was significantly reduced by TNF-α incubation;MLCK significantly increased,while tight junction protein occludin and ZO-1 significantly decreased by TNF-α.These changes were significantly reversed by paeoniflorin,which reduced MLCK expression and enhanced expression of occludin and ZO-1.The protective effects against epithelial barrier dysfunction could be abrogated by small interfering RNA(siRNA) of MLCK.Conclusions Paeoniflorin alleviates the epithelial barrier dysfunction induced by TNF-αthrough down-regulation of MLCK and enhancement of tight junction protein occludin and ZO-1.This study supplies a potential candidate drug for the clinical treatment of inflammatory bowel diseases.
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Objective To investigate the effects of curcumin on myosin light chain kinase(MLCK)in the intestinal mucosa of rats with nonalcoholic fatty liver disease(NAFLD). Methods 35 SD rats were randomly di-vided into the normal control group ,the high fat group and the curcumin group. The high fat group ,curcumin group were given high fat diet for 16 weeks. After 8 weeks of high fat feeding in the curcumin group ,the rats were gavaged with 200 mg/(kg·d)curcumin for 8 weeks. The levels of ALT,AST,LPS and DAO in blood and expres-sion of MLCK in the intestinal mucosa were detected. The changes of liver pathology and tight junction(TJ)of the intestinal mucosa were observed. Results Compared with the control group,the blood levels of ALT,AST,LPS and DAO in the high fat group were obviously increased(P<0.05);Compared with the high fat group,the blood levels of ALT、AST、LPS and DAO in the curcumin group were obviously decreased (P < 0.05). In the high fat group ,hepatocellular steatosis was obvious ,while in the curcumin group hepatocellular steatosis was decreased. TJ was disrupted in the high fat group ,and the intercellular space was larger in the TJ group than the control group (P<0.05). The intercellular space was narrower in the curcumin group than the high fat group(P<0.05). The expression of MLCK in the high fat group was significant higher than that in the control group(P<0.05). The pos-itive staining in the curcumin group was significant lower than that in the high fat group (P < 0.05). Conclusion Curcumin can ameliorate hepatic steatosis by downregulating expression of MLCK in the intestinal mucosa of rats with NAFLD,improving TJ structure of the intestinal mucosa.
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Objective To investigate the effects of curcumin on myosin light chain kinase(MLCK)in the intestinal mucosa of rats with nonalcoholic fatty liver disease(NAFLD). Methods 35 SD rats were randomly di-vided into the normal control group ,the high fat group and the curcumin group. The high fat group ,curcumin group were given high fat diet for 16 weeks. After 8 weeks of high fat feeding in the curcumin group ,the rats were gavaged with 200 mg/(kg·d)curcumin for 8 weeks. The levels of ALT,AST,LPS and DAO in blood and expres-sion of MLCK in the intestinal mucosa were detected. The changes of liver pathology and tight junction(TJ)of the intestinal mucosa were observed. Results Compared with the control group,the blood levels of ALT,AST,LPS and DAO in the high fat group were obviously increased(P<0.05);Compared with the high fat group,the blood levels of ALT、AST、LPS and DAO in the curcumin group were obviously decreased (P < 0.05). In the high fat group ,hepatocellular steatosis was obvious ,while in the curcumin group hepatocellular steatosis was decreased. TJ was disrupted in the high fat group ,and the intercellular space was larger in the TJ group than the control group (P<0.05). The intercellular space was narrower in the curcumin group than the high fat group(P<0.05). The expression of MLCK in the high fat group was significant higher than that in the control group(P<0.05). The pos-itive staining in the curcumin group was significant lower than that in the high fat group (P < 0.05). Conclusion Curcumin can ameliorate hepatic steatosis by downregulating expression of MLCK in the intestinal mucosa of rats with NAFLD,improving TJ structure of the intestinal mucosa.
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Ardipusilloside-I is a natural triterpenoid saponin, which was isolated from Ardisia pusilla A. DC. The aim of the study was to evaluate the stimulation of ardipusilloside-I on gastrointestinal motility in vitro and in vivo. The experiment of smooth muscle contraction directly monitored the contractions of the isolated jejunal segment (IJS) in different contractile states, and the effects of ardipusilloside-I on myosin were measured in the presence of Ca²⁺-calmodulin using the activities of 20 kDa myosin light chain (MLC₂₀) phosphorylation and myosin Mg²⁺-ATPase. The effects of ardipusilloside-I on gastro emptying and intestinal transit in constipation-predominant rats were observed, and the MLCK expression in jejuna of constipated rats was determined by western blot. The results showed that, ardipusilloside-I increased the contractility of IJS in a dose-dependent manner and reversed the low contractile state (LCS) of IJS induced by low Ca²⁺, adrenaline, and atropine respectively. There were synergistic effects on contractivity of IJS between ardipusilloside-I and ACh, high Ca²⁺, and histamine, respectively. Ardipusilloside-I could stimulate the phosphorylation of MLC₂₀ and Mg²⁺-ATPase activities of Ca²⁺- dependent phosphorylated myosin. Ardipusilloside-I also stimulated the gastric emptying and intestinal transit in normal and constipated rats in vivo, respectively, and increased the MLCK expression in the jejuna of constipation-predominant rats. Briefly, the findings demonstrated that ardipusilloside-I could effectively excite gastrointestinal motility in vitro and in vivo.
Subject(s)
Animals , Rats , Ardisia , Atropine , Blotting, Western , Epinephrine , Gastric Emptying , Gastrointestinal Motility , Histamine , In Vitro Techniques , Muscle, Smooth , Myosin Light Chains , Myosin-Light-Chain Kinase , Myosins , Phosphorylation , SaponinsABSTRACT
Objective To explore the impact and mechanism of miR-200 b on intestinal epithelial tight junction. Methods The negative-lentivirus and human-miR-200 b-lentivirus were employed to infect the Caco-2 cell thus establishing two stable cell lines which were then stimulated by 10 ng/mL human tumor necrosis factor-α(TNF-α) to establish the model of the intestinal epithelial injury. Those Caco-2 cells were divided into NC, NC+TNF-α, 200b, and 200b+TNF-αgroups.The tight junction permeability was detected by transepithelial electrical resistance (TEER) and Fluorescein isothiocyanate-labeled dextran (FITC-dextran). The protein alterations myosin light chain kinase (MLCK)/phosphorylated myosin light chain (P-MLC) pathways were measured by Western blot analysis. Results Compared to NC group, NC+TNF-αgroup had lower TEER, higher FITC-dextran, and up-regulated expressions of MLCK and P-MLC proteins (P
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Aim To investigate whether Hcy influenced the intestinal mucosal permeability by regulating MEK-ERK-MLCK pathway. Methods SD rats were divided into 4 groups:normal group, normal+Hcy group, TN-BS/ethanol group, TNBS/ethanol+Hcy group. Experi-mental colitis model with hyperhomocystinemia was es-tablished in rats with intracolonic administration of TN-BS and subcutaneous injection of Hcy. The colonic mucosal tissue was collected for histopathological exam-ination and activity of myeloperoxidase ( MPO ) . The protein expression of MLCK, p-MLCK, MEK, ERK and p-ERK in intestinal mucosal tissues was examined by Western blot method. The mRNA expression of ML-CK was examined by RT-qPCR method. Result Com-pared with the normal group and TNBS group, the DAI and HI scores and the MPO activity were increased in TNBS/ethanol+Hcy group ( P <0. 01 ) . Western blot and RT-qPCR showed that expression of MLCK, p-ML-CK, MEK, ERK and p-ERK increased in small intes-tine in TNBS/ethanol+Hcy group. Conclusion Hcy can increase intestinal permeability in TNBS-induced colitis rats by regulating the expression of MEK-ERK-MLCK signal pathway.
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Objective To study the impact of tumor necrosis factor-α (TNF-α) and its antagonist on the expressions of intestinal mucosa claudin-1,Zonula Occludens-1 (ZO-1) and myosin light chain kinase (MLCK) in rat models of acute liver failure.Methods Fifty four healthy male SpragueDawley (SD) rats were randomly divided into normal control group,model group and intervention group according to a random number table.Rats in normal control (n=6) group were intraperitoneally injected with 0.9％ saline (12 mL/kg).Rats in model group (n=24) and intervention group (n=24) were intraperitoneally injected with a full dose of D-galactosamine (D-GalN) at a dose of 1 200 mg/kg to establish model of acute liver failure,while rats in intervention group were intraperitoneally injected with TNF-α antagonists (rhTNFR∶Fc) at a dose of 12.5 mg/kg before 24 hours given D-GalN.At each time point of hour 8,24,48 and 72,six rats in both model group and the intervention group were sacrificed,respectively,while the normal control group were all anesthetized and sacrificed at 72 h.Models were repeated five times.Serum liver function was detected by biochemical method,and serum TNF-α level was detected by enzyme-linked immunosorbent assay (ELISA).Hematoxylin-eosin (HE) stained sections of liver and terminal ileum were examined under an optical microscope for pathological changes;and protein expression of the terminal ileum Claudin-1,ZO-1 protein and MLCK were determined by immunohistochemistry and Western blot.Means among groups were compared with t test.Results Acute liver failure was successfully induced in the D-GalN injected rats.In the model group,alanine aminotransferase (ALT) began to decline,total bilirubin continued to rise,and enzyme-jaundice separation developed at hour 72.But total bilirubin in intervention group at hour 72 was decreased.Light microscope showed that at hour 72,villus lodged at terminal ileum in the model group with part of villus tip failing off in the model group.Villus mucosa and submucosa interstitial were edema and infiltrated with numerous neutrophils.The terminal ileum kept integrate in the intervention group,and villus mucosa and submucosa were mild edema and only infiltrated with a small amount of neutrophil.Expressions of tumor necrosis necrosis factor (TNF)-α in rats of model group and intervention group were gradually increased and peaked at hour 24 ([239.83 ± 15.81] and [182.71± 17.08] ng/L,respectively),which were significantly higher than that of the control group ([24.19±3.57] ng/L,t=22.68and 15.73,respectively;both P＜0.01).Expression of serumTNF-α in the intervention group was significantly lower than that of model group (t=4.58,P＜0.01).Expressions of Claudin-1 and ZO-1 in model group decreased gradually at an early stage and reached the lowest level at hour 24 (0.355 ± 0.068 and 0.387 ± 0.091,respectively),which were both significantly lower than that of control group (1.640±0.188 and 1.015±0.150,respectively;t=12.87 and 7.14,respectively;both P＜0.01).In the intervention group,expressions of Claudin-1 and ZO-1 also decreased to the lowest level at hour 24 (1.051 ± 0.370 and 0.642 ± 0.082,respectivley),which were both significantly lower than that of control group (t =2.84 and 4.36,respectively;both P＜0.05),but significantly higher than model group with stastically difference (t =3.70 and 4.15,respectively;both P＜0.01).MLCK protein levels in the model and intervention group were gradually increased,which peaked at hour 24 (1.298±0.194 and 1.033 ± 0.073,respectively),significantly higher than the control group (0.460±0.069,t=8.16 and 11.44,both P＜0.01);and MLCK in the intervention group was lower than that in the model group with statistically difference (t=2.56,P＜0.05).Conclusions Expression of serum TNF-α in rat model of acute liver failure increases,which leads to decreased expression of Claudin-1 and ZO-1,and increased expression of MLCK,makes cell shrunk and cell gap increased.TNF-α antagonist could significantly reduce the inflammation and liver cell apoptosis,improve liver function by inhibiting MLCK expression and preventing decrease of Claudin-1 and ZO-1 proteins.