Effect of Intravesical Liposome-Based Nerve Growth Factor Antisense Therapy on Bladder Overactivity and Nociception in a Rat Model of Cystitis Induced by Hydrogen Peroxide
Effect of Intravesical Liposome-Based Nerve Growth Factor Antisense Therapy on Bladder Overactivity and Nociception in a Rat Model of Cystitis Induced by Hydrogen Peroxide
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DOI：
10.1089/hum.2016.121
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发表时间：
2017-07-01
影响因子：
4.2
通讯作者：
Yoshimura, Naoki
中科院分区：
文献类型：
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作者：
Majima, Tsuyoshi;Tyagi, Pradeep;Yoshimura, Naoki
本研究的目的是评估基于脂质体的膀胱神经生长因子（NGF）局部抑制是否对膀胱内灌注过氧化氢（HP）诱导的大鼠膀胱炎模型的膀胱过敏有影响。HP（1.5%）给药于成年雌性Sprague-Dawley大鼠。以TYE563荧光标记的NGF反义寡核苷酸（OND）复合脂质体于第2天静脉滴注。第3天用荧光显微镜观察TYE 563标签的红色荧光。实验采用四组大鼠：(a)假脂质体组，(b)假ond组，(c)囊炎-脂质体组，(d)囊炎- ond组。第0天静脉给予生理盐水或1.5% HP。第2天膀胱内灌注空脂质体或脂质体-反义OND。为评价第7天NGF反义处理的效果，我们进行了以下实验：(a)在清醒状态下进行连续膀胱测定；(b)观察树脂干扰素注入膀胱后引起的疼痛行为，包括舔舐行为（舔下腹部）和冻结行为（不动，头转向下腹部）；(c)膀胱及L6 DRG NGF免疫组化染色；(d)逆转录聚合酶链反应（RT-PCR）分析膀胱中多个基因的表达；(e)将Fast Blue注入膀胱壁上，7天后用激光捕获显微解剖法分别收集DRG神经元内Fast Blue阳性或阴性细胞。RT-PCR检测捕获的神经元细胞中基因表达情况。TYE563仅在尿路上皮表达。在膀胱测量中，与假脂质体组相比，膀胱炎脂质体组的收缩间期（ICI）显著（p = 0.001）缩短。膀胱炎- ond组的ICI明显长于膀胱炎-脂质体组（p = 0.007）。假脂质体组与假ond组比较，ICI无显著差异（p = 0.56）。舔食事件在四组间无显著差异。相比之下，膀胱炎脂质体组明显比假脂质体组出现更多的冻结事件（p = 0.002）。与膀胱炎-脂质体组相比，膀胱炎- ond组的冷冻事件数量显著减少（p = 0.04）。免疫荧光染色显示，膀胱炎-脂质体组粘膜NGF表达（p = 0.02）和L6 DRG表达（p = 0.01）明显高于假脂质体组。与膀胱炎-脂质体组相比，膀胱炎- ond组粘膜中NGF和L6 DRG的表达均显著降低（p = 0.002）。RT-PCR结果显示，膀胱炎性脂质体组黏膜中NGF和TRPV1 mRNA的表达明显高于假脂质体组（p分别为0.001和0.03）。另一方面，这些基因在膀胱炎- ond组的表达明显低于膀胱炎-脂质体组（p分别= 0.007和0.02）。膀胱炎脂质体组标记膀胱传入神经元中TRPA1、P2X3和BDNF mRNA的表达明显高于假脂质体组（p分别为0.03、0.01和0.001）。这些基因在膀胱炎- ond组的表达明显低于膀胱炎-脂质体组（p分别为0.04、0.006和0.03）。本研究表明，脂质体- ngf反义OND膀胱内应用可显著改善大鼠化学性膀胱炎引起的膀胱超敏反应。脂质体- ond结合物膀胱内治疗可能是一种新的局部治疗超敏性膀胱疾病，如膀胱疼痛综合征/间质性膀胱炎。
The aim of this study was to evaluate whether liposome-based local suppression of nerve growth factor (NGF) in the bladder has effects on bladder hypersensitivity in a rat cystitis model induced by intravesical instillation of hydrogen peroxide (HP). HP (1.5%) was intravesically administered to adult female Sprague-Dawley rats. Liposomes complexed with NGF antisense oligonucleotide (OND) labeled with TYE563 fluorescent tag were intravesically instilled on day 2. Red fluorescence from the TYE 563 tag was observed with fluorescent microscopy on day 3. Four separate groups of rats were used in the following experiments: (a) sham-liposome group, (b) sham-OND group, (c) cystitis-liposome group, and (d) cystitis-OND group. Saline or 1.5% HP was intravesically administered on day 0. Empty liposomes or liposomes-antisense OND were instilled into the bladder on day 2. The following experiments were conducted to evaluate the effect of NGF antisense treatment on day 7: (a) continuous cystometry was performed in an awake condition; (b) pain behavior induced by instillation of resiniferatoxin into the bladder, including licking behavior (lower abdominal licking) and freezing behavior (motionless head-turning toward lower abdomen), was observed; (c) immunohistochemical staining of the bladder and L6 DRG for NGF was performed; (d) the expression of several genes in the bladder was analyzed by reverse transcription polymerase chain reaction (RT-PCR); and (e) after Fast Blue was injected into the bladder wall, Fast Blue-positive or -negative cells in DRG neurons were separately collected by using a laser-capture microdissection method 7 days later. RT-PCR was performed to evaluate gene expressions in captured neuronal cells. The expression of TYE563 was identified only in the urothelial layer. In cystometric investigation, intercontraction intervals (ICI) were significantly (p = 0.001) shorter in the cystitis-liposome group in comparison to the sham-liposome group. ICI was significantly (p = 0.007) longer in the cystitis-OND group compared to the cystitis-liposome group. Comparisons of the sham-liposome and the sham-OND groups showed no significant difference in ICI (p = 0.56). Licking events did not significantly differ among the four groups. In contrast, the cystitis-liposome group showed significantly more freezing events than the sham-liposome group did (p = 0.002). A significant reduction in the number of freezing events was observed in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04). Immunofluorescence staining demonstrated that NGF expression in the mucosa (p = 0.02) and L6 DRG (p = 0.01) was significantly higher in the cystitis-liposome group than it was in the sham-liposome group. The expression of NGF was significantly lower in the mucosa (p = 0.002) and L6 DRG (p = 0.01) in the cystitis-OND group compared to the cystitis-liposome group. RT-PCR showed that the expression of NGF and TRPV1 mRNA in the mucosa was significantly higher in the cystitis-liposome group than it was in the sham-liposome group (p = 0.001 and 0.03, respectively). On the other hand, these gene expressions were significantly lower in the cystitis-OND group than they were in the cystitis-liposome group (p = 0.007 and 0.02, respectively). The cystitis-liposome group showed significantly higher expression of TRPA1, P2X3, and BDNF mRNA in labeled bladder afferent neurons than the sham-liposome group did (p = 0.03, 0.01, and 0.001, respectively).These gene expressions were significantly lower in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04, 0.006, and 0.03, respectively). The study indicated that intravesical application of liposome-NGF antisense OND significantly improved bladder hypersensitivity induced by chemical cystitis in rats. Intravesical treatment with liposome-OND conjugates could be a novel local therapy of hypersensitive bladder disorders such as bladder pain syndrome/interstitial cystitis.