从脑肠轴途径探讨隔药饼灸对FGIDs肝郁脾虚模型大鼠胃肠动力障碍和感觉异常影响的作用机制
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摘要
目的：通过观察隔药饼灸对FGIDs肝郁脾虚模型大鼠胃肠吸收、动力和肠道敏感性及脑肠肽CCK、CGRP、Ghrelin、SP表达的影响,从脑肠轴途径探讨隔药饼灸治疗FGIDs的可能的作用机制。
方法：将筛选的60只SD大鼠按随机数字表法随机分为空白组(A)、模型组(B)、隔药饼灸组(C)(药饼由柴胡、茯苓、白术、白芍、陈皮、防风等药组成)、逍遥散组(D)和多潘立酮组(E)5组(12只/组)。A组正常饲养,不做任何干预；其余4组均采用复合病因法(慢性束缚应激+过度疲劳+饮食失节+夹尾30min+摇晃5min)连续造模21d,造模成功后B组捆绑束缚30min,灌服0.9%氯化钠溶液；C组捆绑束缚行隔药饼灸30min,灌服0.9%氯化钠溶液；D组捆绑束缚30min,灌服逍遥散水煎剂；E组捆绑束缚30min,灌服多潘立酮溶液,连续干预治疗14d。治疗结束后,采用间苯三酚法检测尿D-木糖排泄率,采用腹壁撤离反射检测肠道敏感性,并行胃排空、小肠推进实验,取血清和胃肠组织分别采用酶联免疫吸附法和荧光定量PCR法检测CCK、CGRP的表达；取下丘脑、脊髓、胃窦、结肠采用蛋白质印迹法(Western-blot)检测Ghrelin、SP6勺含量。
结果：
1.造模后大鼠表现为扎堆蜷缩在角落、活动减少,神态倦怠,烦躁易怒,毛发枯泽散乱,胡须下垂,目色暗红,眼角留有分泌物,耳廓色淡,吱吱细叫,大便时干时稀；体重增加缓慢；食量下降或增加缓慢；大鼠的穿格数、起立数和修饰数明显减少,大鼠的活动度明显减少；尿D-木糖排泄率持续降低；差异有统计学意义(P<0.05或P<0.01),说明成功复制出了大鼠肝郁脾虚模型。
2.治疗结束后,与A组相比,B组大鼠小肠推进率下降,胃残留率升高,肠道敏感性增加；差异有统计学意义(P<0.05或P<0.01)；与B组相比,C、D、E组大鼠小肠推进率明显升高,胃内残留率明显下降,肠道敏感性降低,差异有统计学意义(P)<0.01),但三组间比较差异无统计学意义(P>0.05)
3.治疗结束后,与A组相比,B组大鼠血清和胃肠组织中CCK的表达降低,CGRP的表达增加,差异有统计学意义(P<0.01)；与B组相比,C、D、E组大鼠血清和胃肠组织中CCK的表达增力,CGRP的表达降低,差异有统计学意义(P<0.05或P<0.01),但三组间比较差异无统计学意义(P>0.05)
4.治疗结束后,与A组相比,B组大鼠下丘脑、脊髓、胃窦和结肠中Ghrelin蛋白含量明显降低,SP物质含量明显升高,差异有统计学意义(P<0.01)；与B组相比,C、D、E组大鼠下丘脑、脊髓、胃窦和结肠中Ghrelin蛋白含量明显升高,SP物质含量明显下降,差异有统计学意义(P<0.01),但三组间比较差异无统计学意义(P>0.05)。
结论：
1.隔药饼灸能明显促进胃的排空、小肠的推进和吸收功能,改善肠道敏感性,其效应与逍遥散和多潘立酮灌服基本相当,其改善胃肠动力障碍和感觉异常的作用可能是隔药饼灸治疗FGIDs勺效应环节之一。
2.隔药饼灸能上调血清、胃肠组织中CCK的表达和下调CGRP的表达水平,其作用与逍遥散和多潘立酮灌服基本相当,其纠正异常的脑肠肽表达的作用可能是隔药饼灸治疗FGIDs6勺关键所在。
3.隔药饼灸能上调下丘脑、脊髓、胃窦和结肠部位的Ghrelin蛋白含量和下调SP的蛋白含量,其作用与逍遥散和多潘立酮灌服基本相当,其调节脑-肠互动异常的作用可能是隔药饼灸治疗FGIDs的重要途径。
总之,本研究使用的隔药饼灸能有效改善胃肠动力障碍和感觉异常,纠正异常的脑肠肽表达,调节脑-肠互动平衡,有助于针灸临床更好地发挥中医辨证论治的特色和优势,提高艾灸与中药外敷治疗的疗效,加之其适应症广、节约药材、操作方便,安全性好,无毒副作用,实用性强,值得进一步临床研究和相关实验研究,并推广运用。
AIM:To observe the influence of Heber-cake-Separated moxibustion on gastrointestinal absorption, gastrointestinal motiliy, the intestinal sensitivity and the brain-gut peptide's expression change of CCK、CGRP、Ghrelin、SP in rats with FGIDs due to syndrome of liver stagnation and spleen deficiency and from the brain-gut axis way to explore the possible mechanism underlying the therapeutic effects of herbal-cake-separated-moxibustion against functional gastrointestinal disorders.
METHODS:Screening of60SD rats were randomly and equally divided into5groups according to the table of random number method:blank group(A), model group(B), herbal-cake-separated moxibustion group(C)(herbal cake is composed of radix bupleuri, poria cocos, atractylodes, radix paeoniae alba, pericarpium citri reticulatae, radix Saposhnikoviae etc), xiao-yao-san group(D) and domperidone group(E),12rats per group. Except for the blank group, the other four groups underwent modeling by applying chron-ic restraint stress+excessive fatigue+irregular food+clip tail for30min+shaking for5min when bounding for21consecutive days. After the success of the building, then Group B was bounded for30min and given0.9%Nacl by ga-vage; Group C was was bounded in underwenting herbal cake separated moxibustion for30min and given0.9%Nacl by ga-vage; Group D was bounded for30min and was given Xiaoyaosan by ga-vage for the same duration, Group E was bounded for30min and was given domperidone by ga-vage, each of them were intervened for14days respectively. After the treatment, D-xylose discharge rate of urine detected by phloroglucinol method, intestinal sensitivity tested by abdominal withdrawal reflex,the gastric emptying、small intestinal transit test were carried out, the expression of CCK, CGRP in serum and gastrointestinal tissues were measured respectively by Elisa and fluorescence quantitative PCR method;the protein content of Ghrelin、SP in hypothalamus、spinal cord、gastric antrum and colon were measured by western blot method.
RESULTS:
1. After the building, the rats were clustering or hiding in the corner, activity decreased, looking tired, being agitated, and the hair withered and scattered mattly, beard prolapsed, eye color darked red, canthus filled with secretion, cochlear color lighted, sounded fine call, loosed stool; the weight was increased slowly; the food intake was decreased or increase slowly, the grid number、stand up number and modified number were significantly reduced the same as the rats'activity; Urine D-xylose discharge rate was continuously reduced in the model rats (P<0.05or P<0.01); all of these illustrated that the model of liver stagnation and spleen deficiency was successful
2. After the treatment,compared with the A group, small intestine transit rate were significantly droped while the gastric residual rate were rised and the intestinal sensitivity were increased significantly in the rats of the B group (P<0.05or P<0.01); compared with B group, small intestine transit rate in the C、 D、Egroup were increased significantly, while the gastric residual rate was dropped and the intestinal sensitivity was reduced significantly(P<0.01), there were no significant differences in the above parameters among the three groups (P>0.05).
3. After the treatment,compared with the A group, the expression of CCK in the serum and gastrointestinal tissues was reduced while the CGRP was increased significantly in the rats of B group,(P<0.01); compared with B group, the expression of CCK in the C、D、E group were increased while the CGRP was decreased significantly(P<0.01or P<0.05), there were no significant differences in the above parameters among the three groups (P>0.05).
4. After the treatment,compared with the A group, the Ghrelin protein content in the hypothalamus, spinal cord, gastric antrum and colon of the model group rats was decreased obviously, while the SP protein content was increased significantly (P<0.01); compared with the B group, the Ghrelin protein content in the hypothalamus, spinal cord, gastric antrum and colon of the C、D、E group was increased significantly,while the SP protein content was decreases obviously (P<0.01), there were no significant differences in the above parameters among the three groups (P>0.05).
CONCLUSION:
1. Herb-cake-Separeted moxibustion can obviously promote the gastric emptying、small intestinal transit and absorption function, improve the intestinal sensitivity the same as xiao-yao-san and domperidone, improving the gastrointestinal motility disorders and paresthesia may be one of the effect links underlying the therapeutic effects of herbal-cake-separated-moxibustion against functional gastrointestinal disorders.
2. Herb-cake-Separeted moxibustion can increase the expression of CCK and decrease the expression of CGRP in the serum and gastrointestinal tissues the same as xiao-yao-san and domperidone, regulating the abnormal brain-gut peptide's expression may be the key to the therapeutic effects of herbal-cake separated-moxibustion against functional gastrointestinal disorders.
3. Herb-cake-Separeted moxibustion can raise the Ghrelin protein content in hypothalamus, spinal cord, gastric antrum and colon parts of and reduce the SP protein content the same as xiao-yao-san and domperidone, adjusting the abnormal of the brain-intestinal interactive may be the important way underlying the therapeutic effects of herbal-cake-separated-moxibustion against functional gastrointestinal disorders.
Anyhow, in this subject, herbal-cake-separated-moxibustion not only can effectively improve gastrointestinal motility disorders and paresthesia, correct the abnormal brain-gut peptide's expression, regulate the balance of the brain-intestinal interactive, but also can help the acupuncture and moxibustion in the clinical to better carry forward the characteristics and advantages in the syndrome differentiation of traditional Chinese medicine, improve the therapeutic effect of moxibustion and traditional Chinese medicine external treatment,combined with wide indication, medicinal material saving, convenient operation,good safety, non-toxic side effects and strong practicability in herbal cake-separated moxibustion, it is worth further clinical research and the related experimental research, and is worth widely using.
方法：将筛选的60只SD大鼠按随机数字表法随机分为空白组(A)、模型组(B)、隔药饼灸组(C)(药饼由柴胡、茯苓、白术、白芍、陈皮、防风等药组成)、逍遥散组(D)和多潘立酮组(E)5组(12只/组)。A组正常饲养,不做任何干预；其余4组均采用复合病因法(慢性束缚应激+过度疲劳+饮食失节+夹尾30min+摇晃5min)连续造模21d,造模成功后B组捆绑束缚30min,灌服0.9%氯化钠溶液；C组捆绑束缚行隔药饼灸30min,灌服0.9%氯化钠溶液；D组捆绑束缚30min,灌服逍遥散水煎剂；E组捆绑束缚30min,灌服多潘立酮溶液,连续干预治疗14d。治疗结束后,采用间苯三酚法检测尿D-木糖排泄率,采用腹壁撤离反射检测肠道敏感性,并行胃排空、小肠推进实验,取血清和胃肠组织分别采用酶联免疫吸附法和荧光定量PCR法检测CCK、CGRP的表达；取下丘脑、脊髓、胃窦、结肠采用蛋白质印迹法(Western-blot)检测Ghrelin、SP6勺含量。
结果：
1.造模后大鼠表现为扎堆蜷缩在角落、活动减少,神态倦怠,烦躁易怒,毛发枯泽散乱,胡须下垂,目色暗红,眼角留有分泌物,耳廓色淡,吱吱细叫,大便时干时稀；体重增加缓慢；食量下降或增加缓慢；大鼠的穿格数、起立数和修饰数明显减少,大鼠的活动度明显减少；尿D-木糖排泄率持续降低；差异有统计学意义(P<0.05或P<0.01),说明成功复制出了大鼠肝郁脾虚模型。
2.治疗结束后,与A组相比,B组大鼠小肠推进率下降,胃残留率升高,肠道敏感性增加；差异有统计学意义(P<0.05或P<0.01)；与B组相比,C、D、E组大鼠小肠推进率明显升高,胃内残留率明显下降,肠道敏感性降低,差异有统计学意义(P)<0.01),但三组间比较差异无统计学意义(P>0.05)
3.治疗结束后,与A组相比,B组大鼠血清和胃肠组织中CCK的表达降低,CGRP的表达增加,差异有统计学意义(P<0.01)；与B组相比,C、D、E组大鼠血清和胃肠组织中CCK的表达增力,CGRP的表达降低,差异有统计学意义(P<0.05或P<0.01),但三组间比较差异无统计学意义(P>0.05)
4.治疗结束后,与A组相比,B组大鼠下丘脑、脊髓、胃窦和结肠中Ghrelin蛋白含量明显降低,SP物质含量明显升高,差异有统计学意义(P<0.01)；与B组相比,C、D、E组大鼠下丘脑、脊髓、胃窦和结肠中Ghrelin蛋白含量明显升高,SP物质含量明显下降,差异有统计学意义(P<0.01),但三组间比较差异无统计学意义(P>0.05)。
结论：
1.隔药饼灸能明显促进胃的排空、小肠的推进和吸收功能,改善肠道敏感性,其效应与逍遥散和多潘立酮灌服基本相当,其改善胃肠动力障碍和感觉异常的作用可能是隔药饼灸治疗FGIDs勺效应环节之一。
2.隔药饼灸能上调血清、胃肠组织中CCK的表达和下调CGRP的表达水平,其作用与逍遥散和多潘立酮灌服基本相当,其纠正异常的脑肠肽表达的作用可能是隔药饼灸治疗FGIDs6勺关键所在。
3.隔药饼灸能上调下丘脑、脊髓、胃窦和结肠部位的Ghrelin蛋白含量和下调SP的蛋白含量,其作用与逍遥散和多潘立酮灌服基本相当,其调节脑-肠互动异常的作用可能是隔药饼灸治疗FGIDs的重要途径。
总之,本研究使用的隔药饼灸能有效改善胃肠动力障碍和感觉异常,纠正异常的脑肠肽表达,调节脑-肠互动平衡,有助于针灸临床更好地发挥中医辨证论治的特色和优势,提高艾灸与中药外敷治疗的疗效,加之其适应症广、节约药材、操作方便,安全性好,无毒副作用,实用性强,值得进一步临床研究和相关实验研究,并推广运用。
AIM:To observe the influence of Heber-cake-Separated moxibustion on gastrointestinal absorption, gastrointestinal motiliy, the intestinal sensitivity and the brain-gut peptide's expression change of CCK、CGRP、Ghrelin、SP in rats with FGIDs due to syndrome of liver stagnation and spleen deficiency and from the brain-gut axis way to explore the possible mechanism underlying the therapeutic effects of herbal-cake-separated-moxibustion against functional gastrointestinal disorders.
METHODS:Screening of60SD rats were randomly and equally divided into5groups according to the table of random number method:blank group(A), model group(B), herbal-cake-separated moxibustion group(C)(herbal cake is composed of radix bupleuri, poria cocos, atractylodes, radix paeoniae alba, pericarpium citri reticulatae, radix Saposhnikoviae etc), xiao-yao-san group(D) and domperidone group(E),12rats per group. Except for the blank group, the other four groups underwent modeling by applying chron-ic restraint stress+excessive fatigue+irregular food+clip tail for30min+shaking for5min when bounding for21consecutive days. After the success of the building, then Group B was bounded for30min and given0.9%Nacl by ga-vage; Group C was was bounded in underwenting herbal cake separated moxibustion for30min and given0.9%Nacl by ga-vage; Group D was bounded for30min and was given Xiaoyaosan by ga-vage for the same duration, Group E was bounded for30min and was given domperidone by ga-vage, each of them were intervened for14days respectively. After the treatment, D-xylose discharge rate of urine detected by phloroglucinol method, intestinal sensitivity tested by abdominal withdrawal reflex,the gastric emptying、small intestinal transit test were carried out, the expression of CCK, CGRP in serum and gastrointestinal tissues were measured respectively by Elisa and fluorescence quantitative PCR method;the protein content of Ghrelin、SP in hypothalamus、spinal cord、gastric antrum and colon were measured by western blot method.
RESULTS:
1. After the building, the rats were clustering or hiding in the corner, activity decreased, looking tired, being agitated, and the hair withered and scattered mattly, beard prolapsed, eye color darked red, canthus filled with secretion, cochlear color lighted, sounded fine call, loosed stool; the weight was increased slowly; the food intake was decreased or increase slowly, the grid number、stand up number and modified number were significantly reduced the same as the rats'activity; Urine D-xylose discharge rate was continuously reduced in the model rats (P<0.05or P<0.01); all of these illustrated that the model of liver stagnation and spleen deficiency was successful
2. After the treatment,compared with the A group, small intestine transit rate were significantly droped while the gastric residual rate were rised and the intestinal sensitivity were increased significantly in the rats of the B group (P<0.05or P<0.01); compared with B group, small intestine transit rate in the C、 D、Egroup were increased significantly, while the gastric residual rate was dropped and the intestinal sensitivity was reduced significantly(P<0.01), there were no significant differences in the above parameters among the three groups (P>0.05).
3. After the treatment,compared with the A group, the expression of CCK in the serum and gastrointestinal tissues was reduced while the CGRP was increased significantly in the rats of B group,(P<0.01); compared with B group, the expression of CCK in the C、D、E group were increased while the CGRP was decreased significantly(P<0.01or P<0.05), there were no significant differences in the above parameters among the three groups (P>0.05).
4. After the treatment,compared with the A group, the Ghrelin protein content in the hypothalamus, spinal cord, gastric antrum and colon of the model group rats was decreased obviously, while the SP protein content was increased significantly (P<0.01); compared with the B group, the Ghrelin protein content in the hypothalamus, spinal cord, gastric antrum and colon of the C、D、E group was increased significantly,while the SP protein content was decreases obviously (P<0.01), there were no significant differences in the above parameters among the three groups (P>0.05).
CONCLUSION:
1. Herb-cake-Separeted moxibustion can obviously promote the gastric emptying、small intestinal transit and absorption function, improve the intestinal sensitivity the same as xiao-yao-san and domperidone, improving the gastrointestinal motility disorders and paresthesia may be one of the effect links underlying the therapeutic effects of herbal-cake-separated-moxibustion against functional gastrointestinal disorders.
2. Herb-cake-Separeted moxibustion can increase the expression of CCK and decrease the expression of CGRP in the serum and gastrointestinal tissues the same as xiao-yao-san and domperidone, regulating the abnormal brain-gut peptide's expression may be the key to the therapeutic effects of herbal-cake separated-moxibustion against functional gastrointestinal disorders.
3. Herb-cake-Separeted moxibustion can raise the Ghrelin protein content in hypothalamus, spinal cord, gastric antrum and colon parts of and reduce the SP protein content the same as xiao-yao-san and domperidone, adjusting the abnormal of the brain-intestinal interactive may be the important way underlying the therapeutic effects of herbal-cake-separated-moxibustion against functional gastrointestinal disorders.
Anyhow, in this subject, herbal-cake-separated-moxibustion not only can effectively improve gastrointestinal motility disorders and paresthesia, correct the abnormal brain-gut peptide's expression, regulate the balance of the brain-intestinal interactive, but also can help the acupuncture and moxibustion in the clinical to better carry forward the characteristics and advantages in the syndrome differentiation of traditional Chinese medicine, improve the therapeutic effect of moxibustion and traditional Chinese medicine external treatment,combined with wide indication, medicinal material saving, convenient operation,good safety, non-toxic side effects and strong practicability in herbal cake-separated moxibustion, it is worth further clinical research and the related experimental research, and is worth widely using.
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