从eHSP释放与TLR信号转导途径探讨艾灸对Hp胃黏膜炎性损伤的干预机制
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摘要
目的：通过观察艾灸对Hp胃炎大鼠胃黏膜损伤指数、胃黏膜HE染色镜检炎症程度评分、eHSP72、TLR2、4受体及受体后下游信号物质的影响,探讨艾灸保护胃粘膜及与eHSP72和TLR2、4免疫信号转导途径各环节之间的相关性,阐明了艾灸保护胃黏膜损伤的免疫学机制,为艾灸预防胃炎和艾灸的“治未病”理论提供试验依据。
方法：本研究分为三个部分。第一部分：60只健康SD大鼠随机分为6组,即A。碳酸氢钠+消炎痛组、A空白组、B模型组、C艾灸组(模型+艾灸穴位组)、D艾灸非穴点组(模型+艾灸非穴点组)和E电针组(模型+电针穴位组),每组10只。采用Hp灌胃建立大鼠Hp胃炎模型,观察胃黏膜损伤指数、Hp快速尿素酶试验、胃黏膜涂片革兰氏染色检查、胃黏膜HE染色镜检炎症程度评分。第二部分：50只健康SD大鼠随机分为5组,即A空白组、B模型组、C艾灸组(模型+艾灸穴位组)、D艾灸非穴点组(模型+艾灸非穴点组)和E电针组(模型+电针穴位组),每组10只。取大鼠血清和胃组织,酶联免疫法检测大鼠血清Hp IgG和eHSP72含量,酶联免疫法检测胃组织匀浆液TNF-α、IL-1β、IL-10、IL-12含量。第三部分：动物分组及处理方法同第二部分。取外周血单核细胞,用实时定量PCR法检测外周血单核细胞TLR2mRNA. TLR4mRNA、CD14mRNA、MyD88mRNA的含量,蛋白免疫印记(Western Bolt)法检测外周血单核细胞NFκB、IκBα的含量。
结果：
1、与A组比较,B组Hp胃炎模型大鼠胃黏膜UI增加,快速尿素酶试验显示呈阳性，胃黏膜涂片革兰氏染色检查检测出Hp，胃黏膜组织HE染色镜检炎症程度评分积分值增高,说明造模成功；与B组比较,C组和E组大鼠胃黏膜UI和胃黏膜组织HE染色镜检炎症程度积分值减低,D组大鼠胃黏膜UI和胃黏膜组织HE染色镜检炎症程度积分值无明显减低；与E组相比,C组大鼠胃黏膜UI值无明显降低(P>0.05)。
2、与A组比较,B组大鼠血清Hp IgG、eHSP72含量明显升高；与B组相比,C组大鼠Hp IgG含量降低,eHSP72含量升高；与D组相比,C组大鼠Hp IgG含量降低,eHSP72含量升高；与E组比较,C组大鼠Hp IgG含量降低,eHSP72含量无明显升高(P>0.05)。
3、与A组比较,B组大鼠胃组织匀浆液TNF-α.IL-1β和IL-12含量升高(P<0.05或P<0.01),胃组织匀浆液IL-10含量降低(P<0.01)；与B组相比,C组大鼠组织匀浆液TNF-α.IL-1β和IL-12含量降低(P<0.01),胃组织匀浆液IL-10含量升高(P<0.01)；与D、E组相比,C组大鼠组织液TNF-α、IL-1β和IL-12含量降低(P<0.05或P<0.01),胃组织匀浆液IL-10含量升高(P<0.01)。
4、与A组比较,B组Hp胃炎大鼠单核细胞TLR2、4mRNA含量增加(P<0.01),CD14mRNA、MyD88mRNA、NFκB含量也增加(P<0.01),IκBα含量降低(P<0.05)；与B组比较,C组大鼠单核细胞TLR2、4mRNA含量降低,CD14mRNA、MyD88mRNA、 NFκB含量也降低(P<0.01),IκBα含量升高(P<0.05)；与D、E组比较,C组大鼠单核细胞TLR2、4mRNA含量降低,CD14mRNA、MyD88mRNA、NFκB含量也降低(P<0.01),IκBα含量升高(P<0.05)。
结论：
1、Hp灌胃造模后可使胃粘膜UI显著增高,胃黏膜组织HE染色镜检炎症程度积分值增高,快速尿素酶试验显示呈阳性,胃黏膜涂片革兰氏染色检查检测出Hp,说明造模成功。
2、艾灸预处理能显著降低Hp胃炎大鼠胃黏膜损伤指数,降低胃黏膜HE染色镜检炎症程度积分,提示艾灸预处理可减轻Hp胃炎大鼠胃黏膜损伤。
3、艾灸预处理能显著增加血清eHSP72含量,降低血清Hp IgG含量,提示艾灸可通过诱导eHSP72的大量表达,启动和调控机体免疫系统对入侵的病原刺激物Hp清除,从而减少Hp IgG的生成。
4、艾灸预处理能显著增加抑炎细胞因子IL-10的含量,降低促炎症细胞因子TNF-α,IL-1β和IL-12的含量,提示艾灸可通过调节抑炎细胞因子和促炎症细胞因子的释放,来干预Hp胃黏膜炎性损伤过程。
5、艾灸预处理能降低外周血单核细胞TLR2、4mRNA、CD14mRNA、MyD88mRNA含量,增加IκBα表达,降低NFκB含量,提示艾灸可通过降低CD14mRNA和TLR2、4mRNA的表达,来调节TLR下游信号转导物质MyD88mRNA含量,增加IκBα表达,抑制NFκB活化,减轻炎症细胞因子的释放。
6、鉴于eHSP72和Hp细胞壁的LPS是TLR受体的内外源配体,eHSP72是机体的免疫“应激信使”,艾灸预处理保护Hp胃黏膜损伤的机制可能与其诱导eHSP72的高表达,通过与TLR2、4受体结合启动受体后信号转导途径,调控下游信号物质的释放,调整机体相关免疫物质的释放,从而清除入侵Hp实现对胃黏膜的保护作用。
7、艾灸和电针穴位组预处理均可降低胃黏膜UI,减轻胃黏膜损伤。相比之下,艾灸组在诱导eHSP72的表达,启动和调控TLR2、4受体及相关信号物质等方面优于电针组。
Objective:To observe the effect on the UI, mucosal microscopy inflammation score after HE dyeing, the expression of serum eHSP72、the content of monocytes TLR2、4mRNA, Toll-like receptor signal transduction substances(MyD88mRNA、 CD14mRNA、IκBa and NFκB)of Hp gastritis in rats with Moxibustion pretreatment, to explore the relationship between the protective effect of gastric mucosal in rats with moxibustion pretreatment and eHSP72、TLR2、4、Toll-like receptor signal transduction pathways,to clarify the immunological mechanisms of protect the gastric mucosa in rats with moxibustion pretreatment,to provide an experimental basis for moxibustion protecting the gastric mucosa and moxibustion prevention from disease.
Methods:The study was divided into three parts. Part one:60healthy SD rats were randomly divided into6groups, namely, NaHCO3and indomethacin group (Group Ao),blank group (Group A), Hp model group(group B), moxibustion at acupoints groups (group C),moxibustion at non-acupoints (group D) and electro-acupuncture acupoints group (group E),10rats in each group. To make gastritis model with Hp gavage, to observe the UI, Hp rapid urease test, gastric mucosal smear gram's stain, the inflammation score of gastric mucosa with HE staining. Part two:50healthy SD rats were randomly divided into5groups, namely, blank group (Group A), Hp model group(group B), moxibustion at acupoints groups (group C),moxibustion at non-acupoints (group D)and electro-acupuncture acupoints group (group E),10rats in each group. To delect the content of serum Hp IgG, eHSP72and TNF-α、IL-1β、IL-10and IL-12in gastric tissue homogenate with enzyme-linked immunosorbent assay. Part three:Groups and processing method are as the same as part two, the content of monocytes TLR2、4mRNA、CD14mRNA、MyD88mRNA were detected by real-time quantitative PCR method, the expression of monocytes NFκB、IκBα wereed detect by western-blot method.
Results:
1. Compared with group A, the UI、the inflammation score of gastric mucosa HE staining were significantly increased in group B, were positive in group B Hp rapid urease test, the Hp were found in gastric mucosal Gram's stain smear; Compared with group B, the UI、the inflammation score of gastric mucosa with HE staining were significantly decreased in group C. There were no significant difference between group C and D in the UI.
2. Compared with group A, the expression of Hp IgG and eHSP72in group B were significantly increased. Compared with group B, the expression of Hp IgG in group B were significantly decreased, the expression of eHSP72in group B were significantly increased. Compared with group D, the expression of Hp IgG in group C were significantly decreased, the expression of eHSP72in group C were significantly increased. Compared with group E, the expression of Hp IgG in group C were significantly decreased, the expression of eHSP72in group C were no significantly increased(P>0.05).
3. Compared with group A, the content of TNF-α、IL-1β、IL-12gastric tissue homogenate were significantly increased (P<0.05或P<0.01), the content of gastric tissue homogenate IL-10were significantly decreased in group B (P<0.01). Compared with group B, the content of TNF-α、IL-1β、IL-12gastric tissue homogenate were significantly decreased (P<0.01), the content of IL-10gastric tissue homogenate were significantly increased in group B (P<0.01). Compared with group D、E, the content of TNF-α、IL-1β、IL-12gastric tissue homogenate were significantly decreased (P<0.05或P<0.01), the content of IL-10gastric tissue homogenate were significantly increased in group B (P<0.01)
4. Compared with group A, the content of monocytes TLR2、4mRNA、CD14mRNA、MyD88mRNA and NFκB were significantly increased (P<0.01), the content of monocytes IκBα were significantly decreased in group B (P<0.05) Compared with group B, the content of monocytes TLR2、4mRNA. CD14mRNA、 MyD88mRNA and NFκB were significantly decreased(P<0.01), the content of monocytes IκBα were significantly increased in group C(P<0.05). Compared with group D、E, the content of monocytes TLR、4mRNA、CD14mRNA MyD88mRNA and NFκB were significantly decreased, the content of monocytes IκBα were significantly increased in group C (P<0.05)
Conclusions:
1. After mading gastritis model with Hp gavage, the UIi、the inflammation score of gastric mucosa with HE staining were significantly increased in rats, Hp rapid urease test were positive, the Hp were found in rats' gastric mucosal Gram's stam smear
2. Moxibustion could decrease the UI of gastritis rats and the inflammation score of gastric mucosa with HE staining, which suggested moxibustion could reduce the Hp gastric mucosal inflammatory injury.
3. Moxibustion could increase the content of eHSP72and decrease the content of Hp IgG, which suggested that moxibustion could induce the expression of a large number of serum eHSP72, start and control the body's immune system to prevent the invasion of pathogen Hp and reduce Hp IgG formation.
4. Moxibustion could increase the content of IL-10and decrease the content of TNF-α、IL-1β、IL-12in gastric tissue homogenate,which suggested moxibustion could adjust the release of proinflammatory cytokine and suppression of inflammatory cytokines, intervene the mucosal inflammatory injury of rats with Hp gastric.
5. Moxibustion could decrease the content of monocytes TLR2、4mRNA、 CD14mRNA、MyD88mRNA, increase the content of monocytes IκBα, decrease the content of monocytes NFκB, which suggested moxibustion could adjust TLR2、4and Toll-like receptor signal transduction substance.
6.The eHSP72and LPS of Hp were endogenous and exogenous ligands of the TLR receptor, eHSP72is the immune stress messenger, moxibustion protecting Hp gastric mucosal injury effect might be related to inducing high the expression of eHSP72, combining eHSP72ligand and TLR2.4receptor, activating and regulating the body immune system of TLR2%4receptor, regulating Toll-like receptor signal transduction substance, adjusting the body's immune substances released to clear the invasion of Hp and protect the gastric mucosa.
7.Moxibustion and electro-acupuncture both could decrease UI, but Moxibustion group in the expression of the eHSP72,TLR2、4receptor, and Toll-like receptor signal transduction substance is better than the electro-acupuncture group.
方法：本研究分为三个部分。第一部分：60只健康SD大鼠随机分为6组,即A。碳酸氢钠+消炎痛组、A空白组、B模型组、C艾灸组(模型+艾灸穴位组)、D艾灸非穴点组(模型+艾灸非穴点组)和E电针组(模型+电针穴位组),每组10只。采用Hp灌胃建立大鼠Hp胃炎模型,观察胃黏膜损伤指数、Hp快速尿素酶试验、胃黏膜涂片革兰氏染色检查、胃黏膜HE染色镜检炎症程度评分。第二部分：50只健康SD大鼠随机分为5组,即A空白组、B模型组、C艾灸组(模型+艾灸穴位组)、D艾灸非穴点组(模型+艾灸非穴点组)和E电针组(模型+电针穴位组),每组10只。取大鼠血清和胃组织,酶联免疫法检测大鼠血清Hp IgG和eHSP72含量,酶联免疫法检测胃组织匀浆液TNF-α、IL-1β、IL-10、IL-12含量。第三部分：动物分组及处理方法同第二部分。取外周血单核细胞,用实时定量PCR法检测外周血单核细胞TLR2mRNA. TLR4mRNA、CD14mRNA、MyD88mRNA的含量,蛋白免疫印记(Western Bolt)法检测外周血单核细胞NFκB、IκBα的含量。
结果：
1、与A组比较,B组Hp胃炎模型大鼠胃黏膜UI增加,快速尿素酶试验显示呈阳性，胃黏膜涂片革兰氏染色检查检测出Hp，胃黏膜组织HE染色镜检炎症程度评分积分值增高,说明造模成功；与B组比较,C组和E组大鼠胃黏膜UI和胃黏膜组织HE染色镜检炎症程度积分值减低,D组大鼠胃黏膜UI和胃黏膜组织HE染色镜检炎症程度积分值无明显减低；与E组相比,C组大鼠胃黏膜UI值无明显降低(P>0.05)。
2、与A组比较,B组大鼠血清Hp IgG、eHSP72含量明显升高；与B组相比,C组大鼠Hp IgG含量降低,eHSP72含量升高；与D组相比,C组大鼠Hp IgG含量降低,eHSP72含量升高；与E组比较,C组大鼠Hp IgG含量降低,eHSP72含量无明显升高(P>0.05)。
3、与A组比较,B组大鼠胃组织匀浆液TNF-α.IL-1β和IL-12含量升高(P<0.05或P<0.01),胃组织匀浆液IL-10含量降低(P<0.01)；与B组相比,C组大鼠组织匀浆液TNF-α.IL-1β和IL-12含量降低(P<0.01),胃组织匀浆液IL-10含量升高(P<0.01)；与D、E组相比,C组大鼠组织液TNF-α、IL-1β和IL-12含量降低(P<0.05或P<0.01),胃组织匀浆液IL-10含量升高(P<0.01)。
4、与A组比较,B组Hp胃炎大鼠单核细胞TLR2、4mRNA含量增加(P<0.01),CD14mRNA、MyD88mRNA、NFκB含量也增加(P<0.01),IκBα含量降低(P<0.05)；与B组比较,C组大鼠单核细胞TLR2、4mRNA含量降低,CD14mRNA、MyD88mRNA、 NFκB含量也降低(P<0.01),IκBα含量升高(P<0.05)；与D、E组比较,C组大鼠单核细胞TLR2、4mRNA含量降低,CD14mRNA、MyD88mRNA、NFκB含量也降低(P<0.01),IκBα含量升高(P<0.05)。
结论：
1、Hp灌胃造模后可使胃粘膜UI显著增高,胃黏膜组织HE染色镜检炎症程度积分值增高,快速尿素酶试验显示呈阳性,胃黏膜涂片革兰氏染色检查检测出Hp,说明造模成功。
2、艾灸预处理能显著降低Hp胃炎大鼠胃黏膜损伤指数,降低胃黏膜HE染色镜检炎症程度积分,提示艾灸预处理可减轻Hp胃炎大鼠胃黏膜损伤。
3、艾灸预处理能显著增加血清eHSP72含量,降低血清Hp IgG含量,提示艾灸可通过诱导eHSP72的大量表达,启动和调控机体免疫系统对入侵的病原刺激物Hp清除,从而减少Hp IgG的生成。
4、艾灸预处理能显著增加抑炎细胞因子IL-10的含量,降低促炎症细胞因子TNF-α,IL-1β和IL-12的含量,提示艾灸可通过调节抑炎细胞因子和促炎症细胞因子的释放,来干预Hp胃黏膜炎性损伤过程。
5、艾灸预处理能降低外周血单核细胞TLR2、4mRNA、CD14mRNA、MyD88mRNA含量,增加IκBα表达,降低NFκB含量,提示艾灸可通过降低CD14mRNA和TLR2、4mRNA的表达,来调节TLR下游信号转导物质MyD88mRNA含量,增加IκBα表达,抑制NFκB活化,减轻炎症细胞因子的释放。
6、鉴于eHSP72和Hp细胞壁的LPS是TLR受体的内外源配体,eHSP72是机体的免疫“应激信使”,艾灸预处理保护Hp胃黏膜损伤的机制可能与其诱导eHSP72的高表达,通过与TLR2、4受体结合启动受体后信号转导途径,调控下游信号物质的释放,调整机体相关免疫物质的释放,从而清除入侵Hp实现对胃黏膜的保护作用。
7、艾灸和电针穴位组预处理均可降低胃黏膜UI,减轻胃黏膜损伤。相比之下,艾灸组在诱导eHSP72的表达,启动和调控TLR2、4受体及相关信号物质等方面优于电针组。
Objective:To observe the effect on the UI, mucosal microscopy inflammation score after HE dyeing, the expression of serum eHSP72、the content of monocytes TLR2、4mRNA, Toll-like receptor signal transduction substances(MyD88mRNA、 CD14mRNA、IκBa and NFκB)of Hp gastritis in rats with Moxibustion pretreatment, to explore the relationship between the protective effect of gastric mucosal in rats with moxibustion pretreatment and eHSP72、TLR2、4、Toll-like receptor signal transduction pathways,to clarify the immunological mechanisms of protect the gastric mucosa in rats with moxibustion pretreatment,to provide an experimental basis for moxibustion protecting the gastric mucosa and moxibustion prevention from disease.
Methods:The study was divided into three parts. Part one:60healthy SD rats were randomly divided into6groups, namely, NaHCO3and indomethacin group (Group Ao),blank group (Group A), Hp model group(group B), moxibustion at acupoints groups (group C),moxibustion at non-acupoints (group D) and electro-acupuncture acupoints group (group E),10rats in each group. To make gastritis model with Hp gavage, to observe the UI, Hp rapid urease test, gastric mucosal smear gram's stain, the inflammation score of gastric mucosa with HE staining. Part two:50healthy SD rats were randomly divided into5groups, namely, blank group (Group A), Hp model group(group B), moxibustion at acupoints groups (group C),moxibustion at non-acupoints (group D)and electro-acupuncture acupoints group (group E),10rats in each group. To delect the content of serum Hp IgG, eHSP72and TNF-α、IL-1β、IL-10and IL-12in gastric tissue homogenate with enzyme-linked immunosorbent assay. Part three:Groups and processing method are as the same as part two, the content of monocytes TLR2、4mRNA、CD14mRNA、MyD88mRNA were detected by real-time quantitative PCR method, the expression of monocytes NFκB、IκBα wereed detect by western-blot method.
Results:
1. Compared with group A, the UI、the inflammation score of gastric mucosa HE staining were significantly increased in group B, were positive in group B Hp rapid urease test, the Hp were found in gastric mucosal Gram's stain smear; Compared with group B, the UI、the inflammation score of gastric mucosa with HE staining were significantly decreased in group C. There were no significant difference between group C and D in the UI.
2. Compared with group A, the expression of Hp IgG and eHSP72in group B were significantly increased. Compared with group B, the expression of Hp IgG in group B were significantly decreased, the expression of eHSP72in group B were significantly increased. Compared with group D, the expression of Hp IgG in group C were significantly decreased, the expression of eHSP72in group C were significantly increased. Compared with group E, the expression of Hp IgG in group C were significantly decreased, the expression of eHSP72in group C were no significantly increased(P>0.05).
3. Compared with group A, the content of TNF-α、IL-1β、IL-12gastric tissue homogenate were significantly increased (P<0.05或P<0.01), the content of gastric tissue homogenate IL-10were significantly decreased in group B (P<0.01). Compared with group B, the content of TNF-α、IL-1β、IL-12gastric tissue homogenate were significantly decreased (P<0.01), the content of IL-10gastric tissue homogenate were significantly increased in group B (P<0.01). Compared with group D、E, the content of TNF-α、IL-1β、IL-12gastric tissue homogenate were significantly decreased (P<0.05或P<0.01), the content of IL-10gastric tissue homogenate were significantly increased in group B (P<0.01)
4. Compared with group A, the content of monocytes TLR2、4mRNA、CD14mRNA、MyD88mRNA and NFκB were significantly increased (P<0.01), the content of monocytes IκBα were significantly decreased in group B (P<0.05) Compared with group B, the content of monocytes TLR2、4mRNA. CD14mRNA、 MyD88mRNA and NFκB were significantly decreased(P<0.01), the content of monocytes IκBα were significantly increased in group C(P<0.05). Compared with group D、E, the content of monocytes TLR、4mRNA、CD14mRNA MyD88mRNA and NFκB were significantly decreased, the content of monocytes IκBα were significantly increased in group C (P<0.05)
Conclusions:
1. After mading gastritis model with Hp gavage, the UIi、the inflammation score of gastric mucosa with HE staining were significantly increased in rats, Hp rapid urease test were positive, the Hp were found in rats' gastric mucosal Gram's stam smear
2. Moxibustion could decrease the UI of gastritis rats and the inflammation score of gastric mucosa with HE staining, which suggested moxibustion could reduce the Hp gastric mucosal inflammatory injury.
3. Moxibustion could increase the content of eHSP72and decrease the content of Hp IgG, which suggested that moxibustion could induce the expression of a large number of serum eHSP72, start and control the body's immune system to prevent the invasion of pathogen Hp and reduce Hp IgG formation.
4. Moxibustion could increase the content of IL-10and decrease the content of TNF-α、IL-1β、IL-12in gastric tissue homogenate,which suggested moxibustion could adjust the release of proinflammatory cytokine and suppression of inflammatory cytokines, intervene the mucosal inflammatory injury of rats with Hp gastric.
5. Moxibustion could decrease the content of monocytes TLR2、4mRNA、 CD14mRNA、MyD88mRNA, increase the content of monocytes IκBα, decrease the content of monocytes NFκB, which suggested moxibustion could adjust TLR2、4and Toll-like receptor signal transduction substance.
6.The eHSP72and LPS of Hp were endogenous and exogenous ligands of the TLR receptor, eHSP72is the immune stress messenger, moxibustion protecting Hp gastric mucosal injury effect might be related to inducing high the expression of eHSP72, combining eHSP72ligand and TLR2.4receptor, activating and regulating the body immune system of TLR2%4receptor, regulating Toll-like receptor signal transduction substance, adjusting the body's immune substances released to clear the invasion of Hp and protect the gastric mucosa.
7.Moxibustion and electro-acupuncture both could decrease UI, but Moxibustion group in the expression of the eHSP72,TLR2、4receptor, and Toll-like receptor signal transduction substance is better than the electro-acupuncture group.
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