Effects of alprostadil in β-aminopropanitrile induced aortic dissection in a murine model / 中华心血管病杂志

Objective: To investigate the protective role of alprostadil on aortic dissection. Methods: 26 C57BL6 male mice were divided into control group (normal drinking water, n=13) and model group (1 g·kg-1·d-1 BAPN via drinking water, n=13). On day 14, mRNA expression of inflammatory-related genes as well as EP receptor families were detected by RT-PCR (n=6 each) and EP4 protein levels were determined by Western blot (n=7 each). Another 88 mice were divided into 3 groups: control group (n=22), model group (n=33) and treatment group (n=33). The mice in model group and treatment group were applied with BAPN (1 g·kg-1·d-1) via drinking water. The mice in treatment group received additional intraperitoneal injection with alprostadil (80 μg·kg-1·d-1) for 28 days. The mice in the control and model group received equal volume intraperitoneal injection with 0.9% saline respectively. The body weight and systolic blood pressure, the mortality and morbidity were monitored from the beginning until the designed end of the study. On day 28, the mice were sacrificed and aorta were fixed, embedded and sliced, followed by staining with HE and Victoria Blue. The distribution of EP4 was determined by immunohistochemistry in control (n=6) and model group (n=6). Furthermore, the concentration of PGE1 were tested among model (n=3) and treatment group (n=4). EP4 protein expression was determined in model group (n=7) and treatment group (n=6). Results: On day 14, mRNA expression level of MCP-1 ((2.74±1.55) vs. (1.00±0.49),<0.05) and MMP2((1.38±0.42) vs. (1.00±0.27), P<0.05) was significantly upregulated in model group compared with control group. Protein expression of EP4 receptor also increased in aorta in model group compared with control group (1.48±0.51 vs. 1.00±0.19, P<0.05). In the dissection area, the EP4 expression was also enriched compared with non-dissection area, particularly in endothelial cells and inflammatory cells on day 28. BAPN applied in drinking water (model and treatment groups) successfully induced the aortic dissection in mice, some mice died of the rupture. The elastic fibers were fractured, and the infiltrated immune cells were visible in dissected tissue. False lumen was formed. There was no dissection and death in the control group. Compared with control group, the morbidity and mortality rates were significantly increased in the model group (60.6%, 20/33, 30.3%, 10/33) and the treatment group (72.7%, 24/33, 24.2%, 8/33). The mortality and morbidity rates were similar between model and treatment groups. There is no difference in terms of SBP among three groups (P>0.05). Further study showed that after alprostadil injection, the blood concentration of PGE1 was increased in treatment group ((0.540±0.041 vs. 0.436±0.012)μmol/L, P<0.05). Besides, the EP4 receptor expression was downregulated in the treatment group compared to model group (0.60±0.30 vs. 1.00±0.20, P<0.05). Conclusion: EP4 expression is upregulated in BAPN induced aortic dissection mouse model. No protective effects are observed post alprostadil treatment in this model probably due to the reduced expression of EP4.

Expression of Prostaglandin E2 Receptors in Acquired Middle Ear Cholesteatoma

OBJECTIVES: To investigate the expression of prostaglandin E2 receptor subtypes, E-prostanoid (EP) 1–4 receptors, in acquired cholesteatoma and its possible role in the pathologic process of this disorder. METHODS: Specimens of human acquired cholesteatoma were obtained from 29 patients and 19 skin biopsies of normal external auditory canal were as controls. The mRNA and protein expression of EP receptors was assessed by quantitative real-time polymerase chain reaction, immunohistochemistry and Western blot. RESULTS: In acquired cholesteatoma, EP1–EP4 receptors were mainly expressed on squamous epithelium and subepithelial infiltrated inflammatory cells. In external auditory canal skin, EP1–EP4 receptors were mainly expressed on squamous epithelium and glandular epithelium. The expression of EP4 receptor on mRNA and protein levels were significant lower in acquired cholesteatoma compared with controls. EP1–EP3 receptors had no significant difference between the experimental and control group. CONCLUSION: Low expression of EP4 may play a crucial role in the pathologic process of inflammation reaction and bone destruction in acquired cholesteatoma, but not EP1, EP2, or EP3 receptors.

Paricalcitol attenuates lipopolysaccharide-induced inflammation and apoptosis in proximal tubular cells through the prostaglandin E₂ receptor EP4

BACKGROUND: Vitamin D is considered to exert a protective effect on various renal diseases but its underlying molecular mechanism remains poorly understood. This study aimed to determine whether paricalcitol attenuates inflammation and apoptosis during lipopolysaccharide (LPS)-induced renal proximal tubular cell injury through the prostaglandin E₂ (PGE₂) receptor EP4. METHODS: Human renal tubular epithelial (HK-2) cells were pretreated with paricalcitol (2 ng/mL) for 1 hour and exposed to LPS (1 μg/mL). The effects of paricalcitol pretreatment in relation to an EP4 blockade using AH-23848 or EP4 small interfering RNA (siRNA) were investigated. RESULTS: The expression of cyclooxygenase-2, PGE₂, and EP4 were significantly increased in LPS-exposed HK-2 cells treated with paricalcitol compared with cells exposed to LPS only. Paricalcitol prevented cell death induced by LPS exposure, and the cotreatment of AH-23848 or EP4 siRNA offset these cell-protective effects. The phosphorylation and nuclear translocation of p65 nuclear factor-kappaB (NF-κB) were decreased and the phosphorylation of Akt was increased in LPS-exposed cells with paricalcitol treatment. AH-23848 or EP4 siRNA inhibited the suppressive effects of paricalcitol on p65 NF-κB nuclear translocation and the activation of Akt. The production of proinflammatory cytokines and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were attenuated by paricalcitol in LPS exposed HK-2 cells. The cotreatment with an EP4 antagonist abolished these anti-inflammatory and antiapoptotic effects. CONCLUSION: EP4 plays a pivotal role in anti-inflammatory and antiapoptotic effects through Akt and NF-κB signaling after paricalcitol pretreatment in LPS-induced renal proximal tubule cell injury.

Inhibitory effect of EP4 antagonist on bone metastasis of prostate cancer / 医学研究生学报

Objective The purpose of this study was to examine the inhibitory effect of ONO-AE3-208, an EP4 antagonist, on prostate cancer with bone metastasis in an animal model . Methods A PC3/LUC cell line was constructed by stably transfecting luciferin to prostate cancer PC 3 cells and inoculated into the left ventricle of nude mice to establish an animal model of prostate cancer with bone metastasis .After modeling , the animals in the experimental group and control groups were intraperitoneally given ONO -AE3-208 and double-distilled water, respectively, followed by examination of the metastasis loci and tumor burden by bioluminescence ima -ging and statistical analysis with survival curves . Results At 60 days after modeling , the animals in the control group exhibited sig-nificantly increased metastases and fluorescence burdens as compared with the experimental group (P<0.01), and the increase was in a time-dependent manner (P<0.01).At 60 days, the controls began to die while the experimental animals remained well alive , and at 180 days, the mice of the control group all died .The survival rate of the animals was significantly higher in the experimental group than in the control ( 13.3% vs 0%, P <0.01 ) and the median survival time remarkably longer in the former than in the latter group (162 d vs 116 d, P <0.01). Conclusion The EP4 antagonist ONO-AE3-208 inhibited the bone metastasis of prostate cancer and prolonged the survival time in the model mice .

Effects of EP4 and EP2 antagonist on the differentiation of Treg/Th17 cells in mice of collagen induced arthritis / 中华风湿病学杂志

Objective To study the effects of EP4 and EP2 antagonists on the differentiation of Treg/ Th17 cells and disease progression in mice of collagen-induced arthritis (CIA) model.Methods DBA/1 mice wereimmunized subcutaneously twice at the root of the tail with type Ⅱ collagen emulsified in Freund's complete adjuvant.EP2 and EP4 antagonist therapies were intraperitoneally administrated for 14 consecutive days after the second immunization.Clinical signs,histological manifestation,serum interleukin (IL)-17 and quantity of CD4+CD25+Foxp3+ Treg cells were determined.ANOVA and t-test were used for statistical analysis.Results Clinical signs of the disease appeared on day 27 and peaked on day 35 after the first immunization.The quantity of CD4+CD25+Foxp3+ Treg cells in spleens [(1.67±0.15)％] and draining inguinal lymph nodes [(3.30±0.36)％] isolated from CIA mice were significantly lower than those of normal DBA/1 mice [(2.77±0.45)％ and (4.73 ±0.45)％ respectively,P＜0.05].Serum IL-17 level of CIA mice [(27±7) pg/ml] was significantly higher than that of normal DBA/1 mice [(14±4) pg/ml,P＜0.05].Intra-peritoneal injection of EP4 but not EP2 antagonist to CIA mice decreased paw edema and swelling,and alleviated the histological manifestations (1.8±1.0 vs 3.5±0.6,P＜0.05) on day 35 after the first immunization.The percentages of CD4+CD25+Foxp3+ Treg cells in both inguinal lymph nodes [(4.20±0.32)％] and spleens [(2.63±0.40)％] were significantly higher in EP4 antagonist-treated but not EP2 antagonist-treated CIA mice compared with CIA mice group [(3.30±0.36)％ and (1.67±0.15)％ respectively,P＜0.05].The level of serum IL-17 was significantly lower in EP4 antagonist-treated [(15±7) pg/ml] but not EP2 antagonist-treated CIA mice compared with CIA mice group [(27±7) pg/ml,P＜0.05].Conclusion EP4 antagonist therapy alleviates clinical symptoms of CIA,improves the histological manifestations,decreases the serum IL-17 level and increases the percentages of CD4+CD25+Foxp3+ Treg cells in both spleens and draining inguinal lymph nodes,so targeting EP4 receptor may be a new possible therapeutic possibility in the prevention and treatment of rheumatoid arthritis.

Possible molecular mechanism of promotion of repair of acute achilles tendon rupture by low intensity-pulsed ultrasound treatment in a rat model / Posible mecanismo molecular de promoción de la ruptura aguda del tendón de aquiles por el tratamiento de ultrasonidos pulsados de baja intensidad en un modelo de rata

OBJECTIVE: This study investigated the effect of Low Intensity-pulsed Ultrasound (LIPUS) on the repair process of ruptured Achilles tendon using a rat model and also examined the regulation of a biological molecule that may contribute to this in vitro and in vitro. METHODS: To investigate the effect of LIPUS and its biological mechanism ofpromoting Achilles tendon repair after acute injury, ninety-eight male Sprague-Dawley (SD) rats (mean body weight, 258 ±9.8 g) aged 12 weeks were used in this study. To create the model, the Achilles tendon attachment site and musculotendinous junction were ruptured under direct vision. The leg on one side was exposed to LIPUS (frequency at 1.5 MHz, the repetition cycle at 1.0 kHz, the burst width at 200 msec and the power output at 45 mW/cm2), for 20 minutes daily with a 0.7 mm diameter probe. Results:Low Intensity-pulsed Ultrasound treatment accelerated the repair of the Achilles tendon compared to the untreated group, judged by electron microscopy. Both cyclo-oxygenase (COX)-2* and EP4* expressions were over-expressed in the LIPUS treated group in the inflammatory period, and TGFJ31* expression was markedly induced in LIPUS treated groups followed by collagen I* and III* expression in the repair and reconstitution process. CONCLUSION: These findings suggest that LIPUS is potentially able to accelerate the repair of acute ruptured Achilles tendon in several ways: by exaggerating inflammation by inducing COX-2 and EP4 and reconstituting tissue by inducing TGFJ31 followed by collagen I and III. (*: p < 0.05, **: 0.001).

OBJETIVO: Este estudio estuvo encaminado a investigar el efecto de los ultrasonidos pulsados de baja intensidad (LIPUS) sobre el proceso de reparación del tendón de Aquiles tras una ruptura, usando un modelo de rata. Asimismo, se examinó la regulación de una molécula biológica que puede contribuir a este proceso in vitro e in vitro. MÉTODOS: Con el fin de investigar el efecto de LIPUS y el mecanismo biológico por el cual este efecto promueve la reparación del tendón de Aquiles tras una lesión aguda, noventa y ocho ratas machos Sprague-Dawley (SD) (peso corporal promedio, 258 ± 9.8 g) de 12 semanas de edad fueron usadas en este estudio. Para crear el modelo, el sitio de ligazón microbiológica del tendón de Aquiles y la unión músculo-tendinosa fueron desgarrados bajo visión directa. La pierna de un lado fue expuesta a LIPUS (frecuencia de 1.5 MHz, ciclo de repetición de 1.0 kHz, ancho de ruptura de 200 msec, y potencia de salida de 45 mW/cm2), por 20 minutos diariamente con una sonda de 0.7 mm diámetro. RESULTADOS: El tratamiento de ultrasonidos pulsados de baja intensidad aceleró la reparación del tendón de Aquiles, en comparación con el grupo no tratado, según se apreció mediante el microscopio electrónico. Tanto la ciclo-oxygenasa (COX)-2* como las expresiones EP4* estuvieron sobe-expresadas en el grupo tratado con LIPUS en el periodo inflamatorio, y la expresión TGFfi1* fue marcadamente inducida en los grupos tratados con LIPUS seguidos por la expresión de colágeno I* y III* en el proceso de reparación y reconstitución. CONCLUSIÓN: Estos resultados sugieren que LIPUS puede potencialmente acelerar la reparación del tendón de Aquiles luego de un desgarramiento, de varias maneras: exagerando la inflamación mediante inducción de COX-2 y EP4 y reconstituyendo el tejido induciendo TGFfil seguido por colágeno I y III. (*: p < 0.05, **: 0.001).

Immunohistochemical Stainning for Ber EP4and EMA in the Diagnosis Basal Cell Carcinoma and Squamous Cell Carcinoma of the Skin / 中华皮肤科杂志

Objective To investigate the significance of immunostainning for Ber EP4and EMA in the diagnosis of basal cell carcinoma and squamous cell carcinoma of the skin.Methods Immunohisto-chemical stainning for Ber EP4and EMA was performed on115cases of basal cell carcinoma,squamous cell carcinoma,Bowen' s disease,actinic keratosis,basosquamous cell carcinoma,seborrheic keratosis,and verruca vulgaris.Specimens were taken from neoplastic tissues as well as the surrounding skin and ap-pendages.Results Ber EP4was positively stained in all cases of basal cell carcinoma and basosquamous cell carcinoma,but negatively stained in squamous cell carcinoma,Bowen's disease,actinic keratosis,sebor-rheic keratosis and verruca vulgaris.Expression of EMA was found in most cases of squamous cell carcinoma and Bowen' s disease,and a few cases of actinic keratosis,and in none of basal cell carcinoma,basosqua-mous cell carcinoma,seborrheic keratosis and verruca vulgaris.Conclusions Routine immunohistochemical staining with both Ber EP4and EMA is helpful for distinction of skin basal cell carcinoma,squamous cell carcinoma,precancerosis and benign hyperplastic dermatoses.