鼻窦炎口服液对急性鼻—鼻窦炎大鼠鼻黏膜β防御素影响的实验研究
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摘要
目的：鼻-鼻窦炎为常见鼻科疾病,发病率较高,属于中医“鼻渊”范畴。熊大经先生立足中医,着眼临床实际,经四十余年的临床凝练,提出其核心学术思想——利枢机、通玄府、和清窍、达膜原,并贯穿于鼻渊的治疗当中。在其治疗鼻渊的基础方——吉雷开窍汤上研制而成的中成药鼻窦炎口服液,经多年临床验证,疗效可靠。我们选用大鼠急性鼻-鼻窦炎动物模型进行实验,观察鼻窦炎口服液对急性鼻-鼻窦大鼠鼻黏膜β-防御素的影响,进一步探讨其治疗鼻渊的机制,结合导师学术思想更好地指导临床用药。
方法：60只SD大鼠随机分为①空白组；②模型组；③鼻窦炎口服液低剂量组；④鼻窦炎口服液中剂量组；⑤鼻窦炎口服液高剂量组；⑥阿莫西林克/拉维酸钾组。急性鼻-鼻窦炎模型依据叶菁报道的方法改良造模。造模7天后,空白组和模型组按5ml·kg-1·d-1灌服无菌水,其余各组分别灌服相应的药物。从造模第1天开始,观察大鼠一般情况变化(体温、体重、局部症状、鼻分泌物等)。用药7天后每组随机选取6只大鼠,测得鼻分泌物pH值；取外周血测得白细胞(WBC)计数与中性粒细胞百分比(N)。之后立即处死大鼠,取得鼻粘膜标本,用免疫组化SP法检测各组大鼠鼻黏膜β-防御素；用RT-PCR法测定大鼠鼻粘膜β-防御素mRNA(BD-2)(?)勺表达；在光学显微镜下观察大鼠鼻黏膜的病理形态变化。
结果：
1.模型组大鼠鼻腔分泌物pH值与空白组相比,显著性升高(P<0.01)。鼻窦炎口服液低剂量组pH值与空白组相比升高,差异有统计学意义(P<0.05)。各治疗组药物均能显著降低鼻腔分泌物pH值(P<0.01)。其中以鼻窦炎口服液高剂量组、阿莫西林/克拉维酸钾组最为明显。
2.急性ARS大鼠WBC计数及N%较空白组明显升高(P<0.01),证明本次实验造模是成功的；各治疗组药物均可使升高的WBC计数下降,其中,鼻窦炎口服药液高剂量组,阿莫西林/克拉维酸钾组效果最明显(P<0.01)。
3.模型组大鼠BD-1平均光密度值(MOD)与空白组相比,无显著性差异(P>0.05)。用药7天后,各治疗组BD-1平均光密度值与模型组、空白组比较,也无显著性差异(P>0.05)。
4.模型组大鼠BD-2平均光密度值(MOD)与空白组相比降低,差异有统计学意义(P<0.05)。用药7天后,除鼻窦炎口服液低剂量组外,各治疗组BD-2平均光密度值较模型组均有所升高,其差异有统计学意义(P<0.05)。各治疗组MOD与空白组相比,无显著性差异(P>0.05)。但是,RT-PCR法测定BD-2mRNA,结果显示各组间BD-2mRNA相对表达量无显著差异(p>0.05)。
5.模型组大鼠BD-3的平均光密度值较空白组升高,其差异有统计学意义(P<0.05)。用药7天,鼻窦炎口服液高剂量组、阿莫西林/克拉维酸钾组BD-3平均光密度值较空白组显著升高,有显著的统计学意义(P<0.01)。各治疗组MOD与模型组相比无显著性差异(P>0.05)。
6.光镜观察显示：模型组大鼠鼻粘膜可见明显的急性炎性病理改变,用药7天后,除鼻窦炎口服液低剂量组,各治疗组药物均使炎性改变减轻,以鼻窦炎口服液高、阿莫西林/克拉维酸钾组效果最明显。
结论：
1.鼻窦炎口服液可以降低ARS大鼠升高的鼻分泌物pH值至正常范围。使得鼻腔内微环境“和谐”。
2.各组大鼠鼻黏膜BD-1的MOD值间没有显著性差异(P>0.05),BD-1可能在大鼠鼻黏膜中呈固有表达。该药物不能诱导BD-1在大鼠鼻黏膜的蛋白表达。
3.模型组大鼠鼻黏膜BD-2蛋白表达较空白组下降(P<0.05),可能与其鼻黏膜组织损伤其功能受到影响,上皮细胞产生的防御素降低有关。鼻窦炎口服液中、高剂量可增强ARS大鼠鼻黏膜BD-2的蛋白表达,发挥防御作用。
4.ARS大鼠鼻黏膜BD-3蛋白表达较空白组增强(P<0.05),BD-3可在炎症条件下被诱导。鼻窦炎口服液高剂量组、阿莫西林/克拉维酸钾组BD-3蛋白表达较空白组显著增强(P<0.01),可能与鼻窦炎口服液高剂量与西药有效的控制局部炎症,鼻黏膜组织修复和功能恢复有关。但是各治疗组与模型组间大鼠鼻黏膜的BD-3蛋白表达水平的差异无统计学意义(P>0.05),尚不能认为鼻窦炎口服液可以影响ARS大鼠鼻黏膜BD-3的蛋白表达。
5.鼻窦炎口服液能有效减轻ARS大鼠局部及全身的炎性改变,其中鼻窦炎口服液高剂量组效果最为明显。
我们推测鼻窦炎口服液以清热利湿启运少阳枢机,宣肺散邪而通玄府、和清窍,扶正祛邪,从整体上调节机体功能,发挥治疗作用。
Objective:Acute rhinosinusitis(ARS) is a common nasal disease with high incidence.My teacher, Mr. Xiong Dajing, who based on the foundation of traditional Chinese medicine, summarized as improving Qi's flowing based on regulating Shao-yang channel, and through opening orifice regulating function of Live and Gall-bladder, tonifying Correct Qi as the core of his cademic thought after more than40years clinical practice, and exerted it throughout his treatment on otorhinolaryngology diseases.Bidouyan-oral liquid (sinusitis oral liquid)which from Ji Lei Kai Qiao Tang has reliable treating effect on allergic.We observe the effect of Bidouyan-oral solution on beta-defensin in nasal mucosa of ARS rats modal,meanwhile, study the mechanism of action about it to Bi Yuan by animal experiment, then to guide the clinical medication more accurately.
Methods:Group the healthy SD rats into the following six parts randomly:blank group, model group, Ji Lei Kai Qiao Tang low-dose group, Ji Lei Kai Qiao Tang middle-dose group, Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group. According to the method reported by YE Jing, we made the Acute Rhinosinusitis model.In the following seven days we drenched the blank group and the model group5ml·kg-1·d-1with the sterile water (SW) for7days, meanwhile the other four groups with corresponding medicine. All these days we observed the changes of the rats about the pH value, body temperature, and body weight, especially the nose symptom and secretory volume. After seven days,6rats randomly choosed in each group to exam ph of nasal secretions, leucocyte count(WBC) and the percent of neutrophils (N%)of peripheral blood cells,then we killed the rats to get nasal mucosa to observe the morphologic changes of it under light microscope. AT the time.SP immunohistochemistry method was used to test BD-1,BD-2,BD-3in nasal mucosa, and RT-PCR(real time polymerase chain reaction) methods was used to test BD-2mRNA.
Results:1.Comparing with the blank group, the pH value of the model group's nasal cavity secretion substance has improved significantly (P<0.01). The nasal cavity secretion substance's pH values of all the therapy groups have declined, especially the Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group.
2.Comparing with the blank group, WBC and N%of the model group has improved significantly (P<.0.01). WBC of all the therapy groups have declined significantly (P<0.01), especially the Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group.
3. MOD for BD-1were no significant difference between all groups(P>0.05).
4.Comparing with the blank group,MOD for BD-2of the model group has declined(P<0.05).After seven days medication, except the Ji Lei Kai Qiao Tang low-dose group, the MOD for BD-2of all the therapy groups have improved(P<0.05) comparing with the model group.But BD-2mRNA were no significant difference between all groups(P>0.05).
5.Comparing with the blank group, MOD for BD-3of the model group has improved(P<0.05).After seven days medication, the MOD for BD-3of the Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group have improved significantly(P<0.01).
6. Using the light microscope,Significant pathological changes of acute inflammation were observed in nasal membrane of the model group,After seven days medication,except the Ji Lei Kai Qiao Tang low-dose group,the nasal membrane of all the therapy groups have returned to normal gradually especially the Ji Lei Kai Qiao Tang high-dose group Amoxicillin and Clavulanate Potassium group.
Conclusion:
1.By falling the Nasal discharge pH value to normal, Bidouyan-oral liquid can make the internal environment concordance of the nasal cavity.
2.MOD for BD-1were no significant difference between all groups(P>0.05).The result suggest that BD-1may paly a constitutive role in nasal defenses.Bidouyan-oral liquid can not induce protein expression of BD-1
3.The middle and high dose Bidouyan-oral liquid can strengthen the protein expression of the nasal mucosa BD-2,to improve the anti-infection ability of the organism.
4.During the acute inflammation, the protein expression of the nasal mucosa BD-3can be strengthened.The result can not suggest that Bidouyan-oral liquid can change protein expression of BD-3
5. Bidouyan-oral liquid can control the general and local inflammatory reaction in ARS rats,especially the high dose.
We suppose that Bidouyan-oral liquid can clear gallbladder and purging heat improve Qi's flowing based on regulating Shao-yang channel,disperse lung-qi and remove evil to open orifice.In this way,it can improve organism internal environment,strengthen vital qi to eliminate pathogenic factor.
方法：60只SD大鼠随机分为①空白组；②模型组；③鼻窦炎口服液低剂量组；④鼻窦炎口服液中剂量组；⑤鼻窦炎口服液高剂量组；⑥阿莫西林克/拉维酸钾组。急性鼻-鼻窦炎模型依据叶菁报道的方法改良造模。造模7天后,空白组和模型组按5ml·kg-1·d-1灌服无菌水,其余各组分别灌服相应的药物。从造模第1天开始,观察大鼠一般情况变化(体温、体重、局部症状、鼻分泌物等)。用药7天后每组随机选取6只大鼠,测得鼻分泌物pH值；取外周血测得白细胞(WBC)计数与中性粒细胞百分比(N)。之后立即处死大鼠,取得鼻粘膜标本,用免疫组化SP法检测各组大鼠鼻黏膜β-防御素；用RT-PCR法测定大鼠鼻粘膜β-防御素mRNA(BD-2)(?)勺表达；在光学显微镜下观察大鼠鼻黏膜的病理形态变化。
结果：
1.模型组大鼠鼻腔分泌物pH值与空白组相比,显著性升高(P<0.01)。鼻窦炎口服液低剂量组pH值与空白组相比升高,差异有统计学意义(P<0.05)。各治疗组药物均能显著降低鼻腔分泌物pH值(P<0.01)。其中以鼻窦炎口服液高剂量组、阿莫西林/克拉维酸钾组最为明显。
2.急性ARS大鼠WBC计数及N%较空白组明显升高(P<0.01),证明本次实验造模是成功的；各治疗组药物均可使升高的WBC计数下降,其中,鼻窦炎口服药液高剂量组,阿莫西林/克拉维酸钾组效果最明显(P<0.01)。
3.模型组大鼠BD-1平均光密度值(MOD)与空白组相比,无显著性差异(P>0.05)。用药7天后,各治疗组BD-1平均光密度值与模型组、空白组比较,也无显著性差异(P>0.05)。
4.模型组大鼠BD-2平均光密度值(MOD)与空白组相比降低,差异有统计学意义(P<0.05)。用药7天后,除鼻窦炎口服液低剂量组外,各治疗组BD-2平均光密度值较模型组均有所升高,其差异有统计学意义(P<0.05)。各治疗组MOD与空白组相比,无显著性差异(P>0.05)。但是,RT-PCR法测定BD-2mRNA,结果显示各组间BD-2mRNA相对表达量无显著差异(p>0.05)。
5.模型组大鼠BD-3的平均光密度值较空白组升高,其差异有统计学意义(P<0.05)。用药7天,鼻窦炎口服液高剂量组、阿莫西林/克拉维酸钾组BD-3平均光密度值较空白组显著升高,有显著的统计学意义(P<0.01)。各治疗组MOD与模型组相比无显著性差异(P>0.05)。
6.光镜观察显示：模型组大鼠鼻粘膜可见明显的急性炎性病理改变,用药7天后,除鼻窦炎口服液低剂量组,各治疗组药物均使炎性改变减轻,以鼻窦炎口服液高、阿莫西林/克拉维酸钾组效果最明显。
结论：
1.鼻窦炎口服液可以降低ARS大鼠升高的鼻分泌物pH值至正常范围。使得鼻腔内微环境“和谐”。
2.各组大鼠鼻黏膜BD-1的MOD值间没有显著性差异(P>0.05),BD-1可能在大鼠鼻黏膜中呈固有表达。该药物不能诱导BD-1在大鼠鼻黏膜的蛋白表达。
3.模型组大鼠鼻黏膜BD-2蛋白表达较空白组下降(P<0.05),可能与其鼻黏膜组织损伤其功能受到影响,上皮细胞产生的防御素降低有关。鼻窦炎口服液中、高剂量可增强ARS大鼠鼻黏膜BD-2的蛋白表达,发挥防御作用。
4.ARS大鼠鼻黏膜BD-3蛋白表达较空白组增强(P<0.05),BD-3可在炎症条件下被诱导。鼻窦炎口服液高剂量组、阿莫西林/克拉维酸钾组BD-3蛋白表达较空白组显著增强(P<0.01),可能与鼻窦炎口服液高剂量与西药有效的控制局部炎症,鼻黏膜组织修复和功能恢复有关。但是各治疗组与模型组间大鼠鼻黏膜的BD-3蛋白表达水平的差异无统计学意义(P>0.05),尚不能认为鼻窦炎口服液可以影响ARS大鼠鼻黏膜BD-3的蛋白表达。
5.鼻窦炎口服液能有效减轻ARS大鼠局部及全身的炎性改变,其中鼻窦炎口服液高剂量组效果最为明显。
我们推测鼻窦炎口服液以清热利湿启运少阳枢机,宣肺散邪而通玄府、和清窍,扶正祛邪,从整体上调节机体功能,发挥治疗作用。
Objective:Acute rhinosinusitis(ARS) is a common nasal disease with high incidence.My teacher, Mr. Xiong Dajing, who based on the foundation of traditional Chinese medicine, summarized as improving Qi's flowing based on regulating Shao-yang channel, and through opening orifice regulating function of Live and Gall-bladder, tonifying Correct Qi as the core of his cademic thought after more than40years clinical practice, and exerted it throughout his treatment on otorhinolaryngology diseases.Bidouyan-oral liquid (sinusitis oral liquid)which from Ji Lei Kai Qiao Tang has reliable treating effect on allergic.We observe the effect of Bidouyan-oral solution on beta-defensin in nasal mucosa of ARS rats modal,meanwhile, study the mechanism of action about it to Bi Yuan by animal experiment, then to guide the clinical medication more accurately.
Methods:Group the healthy SD rats into the following six parts randomly:blank group, model group, Ji Lei Kai Qiao Tang low-dose group, Ji Lei Kai Qiao Tang middle-dose group, Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group. According to the method reported by YE Jing, we made the Acute Rhinosinusitis model.In the following seven days we drenched the blank group and the model group5ml·kg-1·d-1with the sterile water (SW) for7days, meanwhile the other four groups with corresponding medicine. All these days we observed the changes of the rats about the pH value, body temperature, and body weight, especially the nose symptom and secretory volume. After seven days,6rats randomly choosed in each group to exam ph of nasal secretions, leucocyte count(WBC) and the percent of neutrophils (N%)of peripheral blood cells,then we killed the rats to get nasal mucosa to observe the morphologic changes of it under light microscope. AT the time.SP immunohistochemistry method was used to test BD-1,BD-2,BD-3in nasal mucosa, and RT-PCR(real time polymerase chain reaction) methods was used to test BD-2mRNA.
Results:1.Comparing with the blank group, the pH value of the model group's nasal cavity secretion substance has improved significantly (P<0.01). The nasal cavity secretion substance's pH values of all the therapy groups have declined, especially the Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group.
2.Comparing with the blank group, WBC and N%of the model group has improved significantly (P<.0.01). WBC of all the therapy groups have declined significantly (P<0.01), especially the Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group.
3. MOD for BD-1were no significant difference between all groups(P>0.05).
4.Comparing with the blank group,MOD for BD-2of the model group has declined(P<0.05).After seven days medication, except the Ji Lei Kai Qiao Tang low-dose group, the MOD for BD-2of all the therapy groups have improved(P<0.05) comparing with the model group.But BD-2mRNA were no significant difference between all groups(P>0.05).
5.Comparing with the blank group, MOD for BD-3of the model group has improved(P<0.05).After seven days medication, the MOD for BD-3of the Ji Lei Kai Qiao Tang high-dose group and Amoxicillin and Clavulanate Potassium group have improved significantly(P<0.01).
6. Using the light microscope,Significant pathological changes of acute inflammation were observed in nasal membrane of the model group,After seven days medication,except the Ji Lei Kai Qiao Tang low-dose group,the nasal membrane of all the therapy groups have returned to normal gradually especially the Ji Lei Kai Qiao Tang high-dose group Amoxicillin and Clavulanate Potassium group.
Conclusion:
1.By falling the Nasal discharge pH value to normal, Bidouyan-oral liquid can make the internal environment concordance of the nasal cavity.
2.MOD for BD-1were no significant difference between all groups(P>0.05).The result suggest that BD-1may paly a constitutive role in nasal defenses.Bidouyan-oral liquid can not induce protein expression of BD-1
3.The middle and high dose Bidouyan-oral liquid can strengthen the protein expression of the nasal mucosa BD-2,to improve the anti-infection ability of the organism.
4.During the acute inflammation, the protein expression of the nasal mucosa BD-3can be strengthened.The result can not suggest that Bidouyan-oral liquid can change protein expression of BD-3
5. Bidouyan-oral liquid can control the general and local inflammatory reaction in ARS rats,especially the high dose.
We suppose that Bidouyan-oral liquid can clear gallbladder and purging heat improve Qi's flowing based on regulating Shao-yang channel,disperse lung-qi and remove evil to open orifice.In this way,it can improve organism internal environment,strengthen vital qi to eliminate pathogenic factor.
引文
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