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Objective:To explore the correlation between cannabinoid 2 receptor (CB2R) and pyroptosis-related indicators in mice with septic lung injury.Methods:Mice were randomly (ramdon number) divided into four groups ( n=6 per group): sham operation group (sham), mild sepsis group (ALIMi), moderate sepsis group (ALIMo) and severe sepsis group (ALIS). The model of septic lung injury was established by cecal ligation and puncture. The wet-dry weight ratio of lung tissues and lung injury scores were measured 12 hours after operation. The expression of CB2R protein was measured by western blot, and the expression of mRNA of CB2R, NLRP3, caspase-1/11, GSDMD were detected by RT-PCR. Meanwhile ELISA was used to measure the level of inflammatory factor IL-6 and TNF-α. SPSS 22.0 software was used for data analysis. Multiple comparison was analyzed by one-way analysis of variance (one-way ANOVA) and comparison between two groups was performed by LSD test or Games-Howell test. Then, the correlation between the expression of CB2R mRNA and the level of inflammatory cytokines as well as the expression of the pyroptosis-related indicators mRNA was analyzed by pearson correlation analysis, respectively. Results:The statistical value F was obtained by one-way ANOVA and comparison between two groups was performed. Compared to sham group, all above indicators increased with the aggravation of inflammation in the sepsis groups ( P<0.05). Compared to ALIMi group, the concentrations of IL-6 [(277.31±41.07) vs.(140.09±27.56), P<0.05] and TNF-α [(501.09±73.91) vs. (261.36±40.73), P<0.05] in lung tissue homogenate increased in ALIMo group. And the level of CB2mRNA [(2.99±0.28) vs. (2.02±0.19), P<0.05], the expression of CB2 protein [(0.44±0.08) vs.(0.23±0.05), P<0.05] and the level of NLRP3 [(2.53±0.26) vs.(1.61±0.15), P<0.05], caspase-1 [(6.02±0.35) vs.(3.60±0.38), P<0.05], caspase-11 [(11.43±0.83) vs.(6.30±0.65), P<0.05] and GSDMD [(10.46±0.62) vs. (5.67±0.54), P<0.05] mRNA also increased. Compared to ALIMo group, the concentrations of IL-6 [(475.90±67.65) vs. (277.31±41.07), P<0.05] and TNF-α [(713.93±58.85) vs. (501.09±73.91), P<0.05] in lung tissue homogenate increased in ALIS group. And the level of CB2mRNA [(4.00±0.19) vs.(2.99±0.28), P<0.05], the expression of CB2 protein [(0.61±0.05) vs.(0.44±0.08), P<0.05] and the level of NLRP3 [(4.75±0.40) vs.(2.53±0.26), P<0.05], caspase-1 [(8.76±0.72) vs.(6.02±0.35), P<0.05], caspase-11 [(16.31±1.13) vs.(11.43±0.83), P<0.05] and GSDMD [(16.46±1.22) vs. (10.46±0.62), P<0.05] mRNA also increased. Furthermore, correlation analysis showed that there was a highly positive correlation between the expression of CB2R mRNA and the expression of mRNA of NLRP3, caspase-1/11, and GSDMD respectively ( r>0.9, P<0.01). Conclusion:The correlation between the aggravation of inflammation, the indicators of pyroptosis and CB2R mRNA was highly positive in different degrees of septic lung injury. Consequently, CB2R may play a role in the regulatory process of inflammation.
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Objective:To evaluate the relationship between the over-expression of endocannabinoid receptor 2 (CB2R) and macrophage pyroptosis in mice.Methods:Bone marrow-derived macrophages of mice were transfected by lentivirus vector and successfully screened out two stable cell lines: lentivirus LV5 negative control cells (LV5-NC) and lentivirus LV5CB2R overexpressing cells (OE). Two cell lines were respectively divided into 3 groups ( n=18 each) using a random number table method: control group (LV5-NC-C group, OE-C group), LPS/ATP group (LV5-NC-LPS/ATP group, OE-LPS/ATP group) and CB2R specific agonist HU308 group (LV5-NC-HU308 group, OE-HU308 group). Cells in group C were commonly cultured.In LPS/ATP group, cells were incubated with LPS at a final concentration of 0.5 μg/ml for 5 h, and then incubated with ATP at the final concentration of 5 mmol/L for 1 h. In group LPS/ATP+ HU308, cells were incubated for 5 h with LPS at the final concentration of 0.5 μg/ml and HU308 at the final concentration of 10 μmol/L and then with ATP at the final concentration of 5 mmol/L for 1 h. The expression of CB2R, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) mRNA was detected by real-time polymerase chain reaction, the expression of caspase-1 was detected by Western blot, and the concentrations of interleukin-18 (IL-18) and IL-1β in the culture medium were determined by enzyme-linked immunosorbent assay. Results:In each cell line, compared with group C, the expression of NLRP3, caspase-1 and GSDMD was significantly up-regulated, and the concentrations of IL-18 and IL-1β were increased in group LPS/ATP ( P<0.05). Compared with group LPS/ATP, the expression of NLRP3, caspase-1 and GSDMD was significantly down-regulated, the concentrations of IL-18 and IL-lβ were decreased in group HU308 ( P<0.05). There was no significant differences in the indicators mentioned above between group V5-NC-C and group OE-C, between group LV5-NC-LPS/ATP and group OE-LPS/ATP, and between group LV5-NC-HU308 and OE-HU308 ( P>0.05). Conclusion:Over-expression of CB2R gene cannot effectively inhibit the occurrence of macrophage pyroptosis, and only activation of CB2R can inhibit it in mice.
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Objective:To evaluate the role of cannabinoid receptor 2 (CB2R) in macrophage pyroptosis induced by lipopolysaccharide (LPS) in mice.Methods:Macrophage line RAW264.7 cells of mice were routinely cultured and divided into 3 groups ( n=18 each) using a random number table method: control group (group C), LPS group and LPS plus CB2R agonist HU308 group (group HU308). Group C received no mediation, LPS at the final concentration of 1 μg/ml was added in the other groups.After incubation for 15 min, HU308 with the final concentration of 10 μmol/L was added in group LPS+ HU308.All groups were then incubated for 6 h. The expression of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), caspase-1, caspase-11 and gasdermin D (GSDMD) mRNA was measured by real-time polymerase chain reaction, the expression of NLRP3, caspase-1, caspase-11, GSDMD and C-terminal domain of human GSDMD (GSDMD-C) was detected by Western blot, and the concentrations of interleukin-18 (IL-18) and IL-1β in the supernatant were determined by enzyme-linked immunosorbent assay.GSDMD-C/GSDMD ratio was calculated. Results:Compared with group C, the expression of NLRP3, caspase-1, caspase-11, GSDMD and GSDMD-C was significantly up-regulated, GSDMD-C/GSDMD ratio was increased, and the concentrations of IL-18 and IL-1β in the supernatant were increased in group LPS ( P<0.05). Compared with group LPS, the expression of NLRP3, caspase-1, caspase-11, GSDMD and GSDMD-C was significantly down-regulated, GSDMD-C/GSDMD ratio was decreased, and the concentrations of IL-18 and IL-1β were decreased in group HU308 ( P<0.05). Conclusion:CB2R is involved in macrophage pyroptosis induced by LPS in mice.
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Objective To investigate the role of activated cannabinoid receptor 2 (CB2R) in lipopolysaccharide (LPS)-induced secretion of RAW264.7 macrophage inflammatory cytokines and its possible mechanism. Methods Macrophages were seeded in 6-well plates (2 mL/well) at the density of 1×105 cells/mL and randomly divided into four groups (n=6 each group): control group (group C), LPS group (group LPS), LPS plus CB2R agonist HU308 group (group LPS+HU308), and LPS plus HU308 plus 3-Methyladenine group (group LPS+HU308+3-MA). LPS with the final concentration of 1 μg/mL were added in group LPS, group LPS+HU308 and group LPS+HU308+3-MA. After incubation for 15 min, 3-MA with a final concentration of 10 mmol/L was added into group LPS+HU308+3-MA . HU308 with the final concentration of 10 μmol/L was added in group LPS+HU308 and group LPS+HU308+3-MA at 15 min after 3-MA intervention, and the cells were then incubated for 24 h. The concentrations of TNF-α, IL-18 and IL-1β in supernatant serum of each group were determined by ELISA. The expressions of ICAM-1 and NLRP3 mRNA were detected by RT-PCR. The expressions of LC3 and Beclin1 were detected by Western blot, and the ratio of LC3-Ⅱ/LC3-Ⅰ was calculated. LSD-t test was used for sample pairwise comparison, and one way ANOVA for inter-group comparison. A P<0.05 was considered statistically significant. Results Compared with group C, the concentrations of TNF-α [(228.86±10.20) pg/mL vs (140.05±5.54) pg/mL], IL-1β [(363.62±8.14) pg/mL vs (244.82±9.11) pg/mL], and IL-18 [(293.28±13.57) pg/mL vs (202.84±9.54) pg/mL] in supernatant serum were increased (all P<0.05), the expressions of ICAM-1 [(5.88±0.32) vs (1.00±0.03)] and NLRP3 [(8.07±0.93) vs (1.01±0.05)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.50±0.03) vs (0.40±0.06)] and Beclin1 [(0.51±0.04) vs (0.16±0.03)] were up-regulated in group LPS (all P<0.05). Compared with group LPS, the concentrations of TNF-α [(165.44±7.07) pg/mL], IL-1β [(272.09±3.35) pg/mL] and IL-18 [(220.41±6.01) pg/mL] in supernatant serum were significantly decreased (all P<0.05), the expressions of ICAM-1 [(3.21±0.35)] and NLRP3 [(1.54±0.30)] mRNA were decreased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.71±0.03)] and Beclin1 [(0.71±0.02)] were up-regulated in group LPS+HU308 (all P<0.05). Compared with group LPS+HU308, the concentrations of TNF-α [(197.06±5.59) pg/mL], IL-1β [(318.98±11.54) pg/mL] and IL-18 [(243.33±8.71) pg/mL] in supernatant serum were significantly increased (all P<0.05), the expressions of ICAM-1 [(4.04±0.21)] and NLRP3 [(5.87±0.77)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.44±0.08)] and Beclin1 [(0.32±0.03)] were down-regulated in group LPS+HU308+3-MA (all P<0.05). Conclusions Activation of cannabinoid receptor 2 can alleviate LPS-induced the secretion of RAW264.7 macrophage inflammatory cytokines, and its mechanism may be related to enhanced autophagy.
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Objective To evaluate the effect of activating cannabinoid receptor 2 (CB2R) on sepsis-induced acute lung injury and the role of autophagy in mice.Methods Twenty-four SPF male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups (n=6 each) using a random number table method:sham operation group (group Sham),sepsis group (group Sep),sepsis plus CB2R agonist HU308 group (group Sep+HU308) and sepsis plus HU308 plus autophagy inhibitor 3-methyladenine group (group Sep+HU308+3-MA).Sepsis was induced by cecal ligation and puncture in anesthetized mice.HU308 2.5 mg/kg was intraperitoneally injected at 15 min after surgery in Sep+HU308 and Sep+ HU308+3-MA groups,and 15 min later 3-MA 10 mg/kg was intraperitoneally injected in group Sep+ HU308+3-MA.Lung tissues were obtained at 12 h after surgery and stained with haematoxylin and eosin for examination of the pathological changes which were scored and for determination of the expression of tumornecrosis factor-alpha (TNF-α),interleukin-18 (IL-18) and IL-1β mRNA (by real-time polymerase chain reaction),expression of autophagy-related protein 5 (Atg5) (by immuno-histochemistry),and expression of microtubule-associated protein 1 light chain 3 (LC3),Beclin-1 and p62 (by Western blot).The ratio of LC3Ⅱ to LC3Ⅰ (LC3Ⅱ/LC3Ⅰ ratio) was calculated.Results Compared with group Sham,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,and LC3 Ⅱ/LC3 Ⅰ ratio and lung injury score were increased in the other three groups,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep and group Sep+HU308,and the expression of p62 was significantly up-regulated in group Sep+HU308+3-MA (P＜0.05).Compared with group Sep,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly down-regulated,the expression of Atg5 was up-regulated,and lung injury score was decreased in group Sep+ HU308 and group Sep+ HU308 + 3-MA,LC3Ⅱ/LC3Ⅰ ratio was increased,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep+HU308,and the expression of Beclin-1 was down-regulated,and the expression of p62 was up-regulated in group Sep + HU308 + 3-MA (P ＜ 0.05).Compared with group Sep+ HU308,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,the expression of Atg5 and Beclin-1 was down-regulated,LC3Ⅱ/LC3Ⅰ ratio was decreased,the expression of p62 was up-regulated,and lung injury scores were increased in group Sep+HU308+3-MA (P＜0.05).Conclusion Activating CB2R can alleviate acute lung injury in septic mice,and the mechanism may be partially related to enhancing autophagy and reducing inflammatory responses.
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Objective To evaluate the role of M3 receptors in penehyclidine hydrochloride ( PHC)-induced reduction of lipopolysaccharide ( LPS)-induced injury to human pulmonary microvascular endotheli-al cells ( PMVECs) . Methods Human PMVECs transfected with M3 shRNA were seeded in 6-well plates (2 ml∕hole) or in culture flasks (4 ml∕flask) at the density of 1×105 cells∕ml and divided into 5 groups ( n=5 each) using a random number table method: control group ( group C) , LPS group, PHC plus LPS group ( group P+LPS) , LPS plus M3 shRNA transfection group ( group LPS+shRNA) , and PHC plus LPS plus M3 shRNA transfection group ( group P+LPS+shRNA) . Group C received no mediation, and LPS was added at the final concentration of 0. 1 μg∕ml in the other groups. PHC 2 μg∕ml was added at 1 h before adding LPS in P+LPS and P+LPS+shRNA groups. The cells were transfected with plasmid containing 2. 5 nmol∕L M3 receptor shRNA in LPS+shRNA group and P+LPS+shRNA group. Contents of filamentous actin ( F-actin) in endothelial cells were measured by flow cytometry at 1 h after adding LPS. The expression of myosin light chain kinase ( MLCK) and VE-cadherin protein was examined by immunofluorescence. The ex-pression of nuclear factor kappa B ( NF-κB) p65 and IκB was detected by Western blot. Contents of tumor necrosis factor-alpha ( TNF-α) and interleukin-6 ( IL-6) were determined by enzyme-linked immunosorbent assay. The M3 receptor mRNA transcription was detected by real-time polymerase chain reaction at 10, 30 and 60 min after adding LPS. Results Compared with group C, F-actin content was significantly de-creased, the expression of VE-cadherin and IκB was down-regulated, the contents of TNF-αand IL-6 were increased, and the expression of MLCK and NF-κB p65 was up-regulated in LPS and P+LPS groups ( P<0. 05) . Compared with group C, the expression of M3 receptor mRNA was significantly up-regulated in group LPS ( P<0. 05) , and no significant change was found in group P+LPS ( P>0. 05) . Compared with group LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB was up-reg-ulated, the contents of TNF-αand IL-6 were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+LPS and group LPS+shRNA ( P<0. 05) . Compared with group P+LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB protein was up-regulated, TNF-α and IL-6 contents were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+LPS+shRNA ( P<0. 05) . Conclusion PHC re-duces LPS-induced injury to human PMVECs through interfering with M3 receptors and inhibiting NF-κB-mediated inflammatory responses.
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Contrast-enhanced ultrasound (CEUS) is widely used to evaluate tumor microcirculation, which is useful in the differential diagnosis between benignity and malignancy. In the last 10 years, the applicability of CEUS to thyroid nodules has greatly improved due to technological refinements and the development of second-generation contrast agents. In this review, we summarize the applications of CEUS for thyroid nodules, focusing on the imaging findings of malignant and benign nodules in the existing literature and the use of those findings to predict malignancies, with an additional brief description of the utilization of CEUS for other thyroid-related diseases.
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Contrast Media , Diagnosis , Diagnosis, Differential , Elasticity Imaging Techniques , Microcirculation , Thyroid Gland , Thyroid Nodule , UltrasonographyABSTRACT
Objective To investigate the role of M3 receptor in the effect of penehyclidine hydrochloride(PHC) upregulating β-arrestin-1 expression in lipopolysaccharide(LPS)-induced human pulmonary microvascular endothelial cell(HPMVEC) injury.Methods.M3 shRNA transfected HPMVEC and normal HPMVEC cells were randomly divided into LPS group(A),LPS+pHC group(B),LPS+ M3 shRNA transfection group(C) and PHC+ LPS+ M3 shRNA transfection group(D).The cytoskeleton change was observed by laser scanning confocal.The LDH level in cellular supernate was detected.The VCAM 1 protein expression was examined by immunofluorescence chemistry.β-arrestin-1 protein expression was determined by Western blot and β-arrestin-1mRNA expression was measured by real-time PCR.Results Compared with the group A or C,F-actin cytoskeleton arrangement in the group B or D was neat,the LDH level and VCAM-1 protein expression were decreased,and β-arrestin-1 expression was increased;compared with group A or B,F-actin cytoskeleton arrangement in the group C or D was neat,the LDH level and VCAM-1 protein expression were decreased,while the β-arrestin-1 expression had no obvious change.Conclusion Silence M3 receptor is conducive to reduce LPS-induced HPMVEC injury.But the role of PHC up-regulating β-arrestin-1 expression has no necessary connection with M3 receptor.
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Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride (PHC)-induced inhibition of lipopolysaccharide (LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells (PMVECs).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and divided into 5 groups (n=15 each) using a random number table:empty plasmid transfection group (group C),LPS plus empty plasmid transfection group (LPS group),PHC plus LPS plus empty plasmid transfection group (P+LPS group),LPS plus β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group) and PHC plus LPS plus β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).In LPS and LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation,and the cells were then incubated for 1 h.In P+LPS and P+LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,PHC with the final concentration of 2 μg/ml was added at 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation,and the cells were then incubated for 1 h.The cell permeability was measured using Transwell chambers.The expression of heat shock protein (HSP27) was detected by immunofluorescence.The expression of β-arrestin-1,p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK (p-p38MAPK) was detected by Western blot.The ratio of pp38MAPK/p38MAPK was calculated.Results Compared with group C,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in LPS,LPS + shRNA and P + LPS + shRNA groups (P＜0.05),and no significant change was found in the parameters mentioned above in group P+LPS (P＞ 0.05).Compared with group LPS,the cell permeability was significantly decreased,the expression of HSP27 was down-regulated,p-p38MAPK/p38MAPK ratio was decreased,and the expression of β-arrestin1 was up-regulated in group P +LPS,and p-p38MAPK/p38MAPK ratio was significantly increased (P＜0.05),and no significant change was found in the other parameters in group P+LPS+shRNA (P＞0.05).Compared with group P+LPS,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs.
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Objective To evaluate the effect of continuous low-to medium-flow oxygen administration in non-ventilated lung on the oxidative stress response of lung tissues during one-lung ventilation (OLV).Methods Fifty-seven American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients,aged 50-64 yr,weighing 40-74 kg,scheduled for elective pulmonary tumorectomy,were divided into 2 groups using a random number table:control group (group C,n=29) and continuous oxygen administration group (group O,n =28).The patients were intubated with the double-lumen tube after induction of anesthesia.Correct position of the tube was verified with the fiberoptic bronchoscope.In group O,the F14 tube was placed at 2-3 cm beyond the carina of trachea in the non-ventilated lung at the beginning of OLV,and low-to medium-flow oxygen was continuously administered at 1-4 L/min with the fractional concentration of inspired oxygen set at 25％-37％.Blood samples were taken from the radial artery and internal jugular bulb at the beginning of anesthesia induction (T1) and 30 min,1 h and 2 h of OLV (T2-4) for blood gas analysis.Lung tissues at the site 5 cm lateral to the tumor were taken immediately after resection of diseased tissues for determination of superoxide dismutase and malondialdehyde levels (by chemical colorimetry) and heme oxygenase-1 expression (by Western blot).Results Compared with group C,the partial pressure of arterial oxygen was significantly increased at T2-4,the partial pressure of arterial carbon dioxide was decreased at T2,3,the partial pressure of venous oxygen was increased at T2,3,the partial pressure of venous carbon dioxide was decreased at T2-4,the malondialdehyde level was decreased,and the expression of heme oxygenase-1 was up-regulated (P＜0.05),and no significant change was found in superoxide dismutase level in group O (P＞0.05).Conclusion The mechanism by which continuous low-to medium-flow oxygen administration in non-ventilated lung exerts pulmonary protection is related to inhibiting oxidative stress responses of lung tissues during OLV.
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Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells (PMVECs) caused by endotoxin and the relationship with mitogen-activated protein kinase (MAPK) signaling pathway.Methods Human PMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and randomly divided into 6 groups (n=5 each):control group (group C),M3 receptor shRNA transfection group (group shRNA),lipopolysaccharide (LPS) group,penehyclidine plus LPS group (group P+LPS),LPS plus M3 receptor shRNA transfection group (group LPS+shRNA) and PHC plus LPS plus M3 shRNA transfection group (group P+LPS+shRNA).The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA,LPS+shRNA and P+LPS+shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation,cells were incubated for 1 h,then LPS at the final concentration of 0.1 μg/ml was added,and cells were incubated for another l h in P+LPS and P+LPS+shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) was detected by Western blot,the expression of heat shock protein 27 (HSP27) using immunofluorescent staining,and the expression of M3receptor mRNA by real-time polymerase chain reaction.Results Compared with group C,M3 receptor mRNA expression was significantly down-regulated in group shRNA,and the permeability of cells was significantly increased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was up-regulated in group LPS (P＜0.05).The permeability of cells was significantly decreased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was down-regulated in P+ LPS,LPS+shRNA and P+LPS+shRNA groups as compared with group LPS,and in group P+LPS+shRNA as compared with group LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.
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Objective To evaluate the effect of dexmnedetomidine pretreatment on apoptosis in hippocampal neurons of rats undergoing one-lung ventilation (OLV).Methods Ninety adult male SpragueDawley rats,aged 10-11 months,weighing 260-300 g,were divided into 3 groups (n=30 each) using a random number table:two-lung ventilation (TLV) group,group OLV and dexmedetomidine pretreatment group (group D).Dexmedetomidine 25 μg/kg was injected intraperitoneally at 45 min before OLV in group D.After tracheal intubation,the animals were ventilated in volume-controlled mode.OLV was performed for 90 mnin followed by 30 min of TLV in OLV and D groups.TLV was performed for 120 min in group TLV.On 1,3 and 7 days after ventilation,6 rats in each group were selected,and Morris water maze test was carried out to evaluate the cognitive function.The swimming speed,time of staying at the target quadrant,and frequency of crossing the platform quadrant were recorded.Six rats in each group were selected immediately after ventilation and sacrificed,the hippocampi were removed for detection of cell apoptosis,and the apoptosis index was calculated.Immediately after ventilation and on 1,3 and 7 days after ventilation,6 rats in each group were selected and sacrificed,and the hippocampi were removed for determination of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2),phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB),Bcl-2 and Bax expression.The ratio of Bcl-2 expression to Bax expression (Bcl-2/Bax ratio) was calculated.Results Compared with group TLV,the time of staying at the target quadrant was significantly shortened,the frequency of crossing the platform quadrant was decreased,the apoptosis index was increased,the expression of pERK1,pERK2,pCREB and Bcl-2 was down-regulated,the expression of Bax was up-regulated,and Bcl-2/Bax ratio was decreased in group OLV (P＜0.05 or 0.01).Compared with group OLV,the time of staying at the target quadrant was significantly prolonged,the frequency of crossing the platform quadrant was increased,the apoptosis index was decreased,the expression of pERK1,pERK2,pCREB and Bcl-2 was up-regulated,the expression of Bax was down-regulated,and Bcl-2/Bax ratio was increased in group D (P＜0.05 or 0.01).Conclusion Dexmedetomidine pretreatment decreases apoptosis in hippocampal neurons through activating ERK/CREB signaling pathway,thus reducing cognitive dysfunction of rats undergoing OLV.
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Objective To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml,and randomly divided into 5 groups (n=20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (LPS group),PHC + LPS + empty plasmid transfection group (P + LPS group),LPS+β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group),and PHC + LPS+β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,the cells were incubated for 24 h.At 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added,and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+LPS and P+LPS+shRNA groups,PHC with the final concentration of 2 μg/ml was added,and the cells were incubated for 1 h,and then LPS with the final concentration of 0.1 μg/ml was added,and the cells were incubated for 1 h.The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated cJun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction.Results Compared with group C,the expression of Factin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group LPS,and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P＜0.05),and no significant change was found in the other parameters mentioned above in group P+LPS (P＞0.05).Compared with group LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was down-regulated in group P+LPS,and the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK and p-JNK was up-regulated in group LPS+shRNA (P＜0.05).Compared with group P+LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression.
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Objective To evaluate the role of β-arrestin-1 in inhibition of endotoxin-induced activation of nuclear factor kappa B (NF-κB) in human pulmonary microvascular endothelial cells (HPM-VECs) by penehyclidine hydrochloride (PHC).Methods HPMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105/ml,and were randomly divided into 5 groups (n =20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (group LPS),PHC + LPS + empty plasmid transfection group (group P+LPS),LPS + β-arrestin-1 gene-shRNA transfection group (group LPS+shRNA) and PHC + LPS + β-arrestin-1 gene-shRNA transfection group (group P+LPS+shRNA).HPMVECs were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1gene-shRNA.At 24 h of incubation,PHC with the final concentration of 2 μg/ml was added,the cells were incubated for 1 h,LPS with the final concentration of 0.1 μg/ml was then added,and the cells were continuously incubated for another 1 h.The supernatant was collected to measure the activity of lactic dehydrogenase (LDH).The cell suspension was collected for determination of vascular cell adhesion molecule-1 (VCAM-1) expression and NF-κB activities and NF-κB inhibitor I-κB and β-arrestin-1expression.Results Compared with group C,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in LPS and LPS+shRNA groups.Compared with group LPS,the activities of LDH in supernatant were decreased,VCAM-1 expression was down-regulated,NF-κB activity was significantly decreased,and I-κB and β-arrestin-l expression was up-regulated in group P+LPS,and no significant change was found in the parameters mentioned above in group P+LPS+shRNA.Compared with group P+LPS,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in group P+LPS+shRNA.Conclusion PHC inhibits endotoxin-induced activation of NF-κB in HPMVECs completely through up-regulating β-arrestin-1 expression.
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Objective To assess the value of early evaluation of left ventricular global systolic function in patients with metabolic syndrome(MS) by three-dimensional speckle tracking echocardiography (3D-STI).Methods 33 healthy subjects and 41 MS subjects who didn't have the left ventricular remodeling were recruited in this study,and the myocardial motions were tracking by 3D-STI.The parameters of left ventricular global longitudinal peak systolic strain(LS),circular peak systolic strain(CS),radial peak systolic strain(RS) and area peak systolic strain (AS) and the values of body mass index (BMI),waist circumference,waist-to-hip ratio and biochemical indicators were compared between the two groups.Results Compared with controls,LS of MS group was significantly decreased (P ＜ 0.01),while no significantly differences were found in CS,RS,and AS(P ＞0.05).The BMI,waist circumference and waist-to-hip ratio were positive correlated with LS (r =0.559,0.617,0.681,P ＜0.01).Conclusions 3D-STI could early evaluate the changes of left ventricular global systolic function for metabolic syndrome patients,and longitudinal peak systolic strain could be the most sensitive index.
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Objective To investigate the effects of penehyclidine hydrochloride (PHCD) pretreatment on β-arrestin-1 expression during sepsis-induced acute lung injury in mice.Methods Thirty female Kunming mice,weighing 18-20 g,were randomly divided into 3 groups (n =10 each):sham operation group (S group),sepsis group (CLP group) and PHCD group.Sepsis was induced by cecal ligation and puncture (CLP).In PHCD group,PHCD 0.45 mg/kg was injected intraperitoneally 1 h before CLP.The equal volume of normal saline was given instead in groups S and CLP.The mice were sacrificed at 12 h after CLP,bronchoalveolar lavage fluid (BALF) was collected for measurement of the total protein concentration,and the lungs were removed for determination of wet/dry lung weight ratio and expression of myosin light chain kinase (MLCK),vascular endothelial cadherin (VE-cad-herin) and β-arrestin-1 in lung tissues.The pathological changes of the lung were scored.Results Compared with group S,the lung injury score,wet/dry lung weight ratio and total protein concentration in BALF were significantly increased,MLCK expression was up-regulated and VE-cadherin expression was down-regulated in groups CLP and PHCD,β-arrestin-1 expression was down-regulated in group CLP and β-arrestin-1 expression was up-regulated in group PHCD (P ＜ 0.05 or 0.01).The lung injury score,wet/dry lung weight ratio,total protein concentration in BALF,and MLCK expression were significantly lower,while the expression of VE-cadherin and β-arrestin-1 was higher in PHCD group than in CLP group (P ＜ 0.05 or 0.01).Conclusion PHCD pretreatment can ameliorate acute lung injury through up-regulating β-arrestin-1 expression and reducing microvascular permeability in septic mice.
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Objective To investigate the effects of penehyclidine hydrochloric(PHC)pretreatment on the expression of β-arrestin-2 in the lung tissue in sepsis-induced acute lung injury in mice.Methods Thirty female Ktmming mice,aged 6 weeks,weighing 18-20 g,were randomly divided into 3 groups(n =10 each):sham operation group(group S); sepsis group(group CLP)and penehyclidine hydrochloric pretreatment group(group PHC).Sepsis was induced by cecal ligation and puncture(CLP)in groups CLP and PHC.Penehyclidine hydrochloric 0.45 mg/kg was injected intraperitoneally at 1 h before CLP in group PHC.While the equal volume of normal saline was given instead of penehyclidine hydrochloric in groups S and CLP.At 12 h of CLP,the animals were sacrificed,and the lung tissues were removed for determination of MPO activity(by colorimetry),IL-6 content(by ELISA),β-arrestin-2 mRNA and protein expression(by RT-PCR and Western blot respectively).Blood samples and bronchoalveolar lavage fluid were collected to calculate pulmonary vascular permeability index(PV PI).Results Compared with group S,PVPI,IL-6 content and MPO activity were significantly increased,the expression of β-arrcstin-2 protein was significantly down-regulaled while the expression of β-arrestin-2 mRNA was up-regulated in group CLP,and PVPI,IL-6 content and MPO activity were significantly incrcased,the expression of β-arrestin-2 protein was significantly up-regulated,while the expression of β-arrestin-2 mRNA was down-regulated in group PHC(P ＜ 0.05).Compared with group CLP,PVPI,IL-6 content,and MPO activity were significantly decreased,the expression of β-arrestin-2 protein was significantly up-regulated,while the expression of β-arrestin-2 mRNA was dow n-regulated in group PHC(P ＜ 0.05).Conclusion PHC pretreatment can attenuate the lung injury induced by sepsis in mice through up-regulating the expression of β-arrestin-2 protein.
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Objective To investigate the effects of galantamine on the myocardial ischemia-reperfusion (I/R) injury in rats and the possible mechanism.Methods Fifty male SD rats weighing 225-275 g were randomly assigned into 5 groups (n =10 each):sham operation group (group SH); I/R group; galantamine + I/R group (group GAL); M receptor antagonist atropine + galantamine + I/R group (group AT); vagus nerve cut-off + galantamine + I/R group (group VGT).Myocardial I/R was induced by occlusion of left anterior descending branch of coronary artery for 30 min followed by 120 min reperfusion.Normal saline 2 ml/kg was slowly injected via the femoral vein at 30 min before ischemia in groups SH and I/R.Galantamine 4 mg/kg was slowly injected via the femoral vein at 30 min before ischemia in group GAL.Atropine 4 mg/kg was slowly injected via the femoral vein at 45 min before ischemia in group AT and the other procedures were the same as those in group GAL.Bilateral cervical vagus nerves were cut off at 45 min before ischemia in group VGT and the other procedures were the same as those in group GAL.At the end of reperfusion,the hearts were removed for determination of myocardial infarct size,MPO and SOD activities,and MDA contents.Results The myocardial infarct size was significantly larger,the MPO activity and MDA content were significantly higher,and the SOD activity was significantly lower in group I/R than in group SH,and in groups AT and VGT than in group GAL (P ＜ 0.05).The myocardial infarct size was significantly smaller,the MDA content and MPO activity were significantly lower,and the SOD activity was significantly higher in group GAL than in group I/R P ＜ 0.05).Conclusion Galantamine has protective effect on myocardium against I/R injury and regulation of peripheral vagus nerve tension may be involved in the mechanism.
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Objective To investigate the role of p38 mitogen-activated protein kinase (p38 MAPK) in attenuation of lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell injury by penehyclidine hydrochloride (PHC).Methods Human umbilical vein endothelial cells were provided by Medical Research Center,Wuhan University,cultured and seeded in 96-well plate (100 μl/hole) or 24-well plate (3 nl/hole) with density of 1 × 104/ml or in culture flasks (5 ml/flask) with density of 1 × 106/ml.The cells were randomly divided into 4 groups ( n =23 each):group control (group C) ; group LPS; group PHC (group P) and group PHC + LPS (group PL).The cells were exposed to LPS 1 μg/ml in groups L and PL or/and PHC 2 μg/ml in groups P and PL.LPS was added at 1 h after PHC in group PL.The cells were collected at 24 h exposure to LPS for determination of the expression of phosphorylated p38 MAPK (p-p38 MAPK) and p38 MAPK.The ratio between p-p38 MAPK and p38 MAPK was calculated.Cell viability,NO content and inducible nitric oxide synthase (iNOS) expression were also determined.Results LPS significantly decreased cell viability,increased NO content,iNOS expression,p-p38 MAPK and p-p38 MAPK/p38 MAPK ratio in group L as compared with group C.In group PL pretreatment with PHC significantly attenuated LPS-induced cell injury.Conclusion p38 MAPK pathway is involved in attenuation of LPS-induced endothelial cell injury by PHC.
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Objective To investigate the effect of penehyclidine hydrochloride (PHCD) on tramadol dependence and c-fos,△ FosB and M5 receptor expression in relevant brain regions in rats.Methods Thirty male adult SD rats weighing 180-220 g were randomly assigned to 3 groups (n =10 each):control group (group C),tramadol dependence group (group T) and PHCD group (group P).Tramadol dependence was induced by subcutaneous 10 mg/kg once a day for 7 consecutive days in groups T and P.PHCD 1.5 mg/kg was injected intraperitoneally on day 8 in group P,while in groups C and T the equal volume of normal saline was injected intraperitoneally instead of PHCD.The rats underwent conditioned place perference test at 30 min after intraperitoneal injection.The time spent in drug-paired side (gray area) was recorded.The rats were sacrificed after the conditioned place perference testand the brain was removed.The relevant brain regions (ventral tegmental area,prefrontal cortex,nucleus accumbens )were separated for determination of c-fos,△ FosB expression by Western blot and M5 receptor mRNA expression by RT-PCR.Results Compared with group C,the time spent in the drug-paired side (gray area) was significantly prolonged,and c-fos,△FosB and M5 receptor mRNA expressions were up-regulated in group T,△FosB and Ms receptor mRNA expressions were down-regulated in group P ( P ＜ 0.05 or 0.01 ).There was no significant difference in time spent in the drug-paired side (gray area) and c-fos expression between groups C and P( P ＞ 0.05).Compared with group T,the time spent in the drug-paired side (gray area) was significantly shortened,and c-fos,△ FosB and M5 receptor mRNA expressions were down-regulated in group P (P ＜0.01).Conclusion PHCD can significantly inhibit tramadol dependence by down-regulating c-fos,△FosB and M5 receptor expression in relevant brain regions.