Efficacy of herb-partitioned moxibustion at Qihai(CV 6)and bilateral Tianshu(ST 25)on colonic damage and the TLR4/NF-κB signaling pathway in rats with Crohns disease

INTRODUCTION

39 Kawasaki T,Kawai T.Toll-like receptor signaling pathways.Front Immunol 2014;5:461.

Some complementary and alternative therapies for CD have been applied,with the aim of improving gastrointestinal function and quality of life.Studies have shown that acupuncture and moxibustion have notable advantages in relieving the symptoms of CD patients,improving quality of life,and alleviating the side effects caused by conventional western medicines.5,6Herb-partitioned moxibustion(HPM)has a long history of being used to prevent or treat diseases via fumigating and warming the acupoints with an ignited moxa cone or moxa roll.It has been proved that moxibustion at Tianshu(ST 25)and Qihai(CV 6)has significant efficacy in treating mild to moderate CD.7-9However,the action mechanism of moxibustion is still unclear,which limits its worldwide clinical application.

The fundamental characteristic of CD is nonspecific colonic inflammation;10,11a great numberof abnormally activated immunocytes are assembled in the colon,while variousinflammatory factorsare overexpressed.12,13Toll-like receptor 4(TLR4),one of the key proteins involved in nuclear factor κB(NF-κB)transduction,is highly expressed in the colonic mucous of CD patients.14,15Our previous study implied that HPM can regulate the expression of miRNA-147,which mainly regulatesthe TLR4/NF-κB signaling pathway.16,17Therefore,the present study aimed to observe the effects of HPM on colonic damage and TLR4/NF-κB signaling pathway transduction in a rat model of CD induced by trinitrobenzenesulfonic acid(TNBS),and to investigate the possible action mechanism of HPM.

MATERIALS AND METHODS

Animals

Forty Sprague-Dawley male rats of clean grade and weight[(150±10)g]were provided by the Experimental Animal Center of the Medical College of Fudan University [certificate No. SCXX (Shanghai)2009-0019].Feeding conditions were:clean grade,room temperature18-22℃,relativehumidity 50%-70%,with feed and water provided ad libitum.After 3 d of adaption,a random number table was used to divide the rats equally into four groups:normal control(NC),model control(MC),mesalamine medication(MES),and HPM.This study was carried out in strict accordance with the guidelines provided by the National Institutes of Health for the Care and Use of Laboratory Animals.The animal protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University.All surgery was performed under sodium pentobarbital anesthesia,and all efforts were made to minimize suffering.

可能原因是高职学生的年龄、文化修养、自我认同等因素。进一步分析中职生和高职生各维度的相关因素发现,中职和高职组在把握感和一致感维度有显著性差异(见表4)。

Crohn's disease model preparation

It is currently considered that immunoregulatory abnormalities are involved in the occurrence and development of CD.Mesalamine is a type of 5-aminosalicylic acid medicine that is commonly used to treat mild to moderate CD.22,23The mechanisms of mesalamine in treating CD include inhibition of natural killer cell activity,antibody formation,formation of leukotriene and prostaglandin-like materials,and scavenging of free oxygenradicals.24,25Mesalamine is also effective in reducing inflammation in IBD by influencing the activation of NF-κB.26,27However,the European Crohn's Disease and Colitis Organization discourages the use of 5-aminosalicylic acid for the treatment of active CD.28,29Furthermore,many studies have confirmed the clinical limitations of mesalamine in treating CD.For example,hormones or other medicines are suggested as alternatives when mesalamine provides no improvement after 4-8 weeks,which is probably due to its fractional effect and failure to reach deep intestinal lesions as a local anti-inflammatory agent.30In addition,intolerance to mesalamine is reportedly experienced by up to 15%of patients,including diarrhea in 3%,headache in 2%,nausea in 2%,and rash in 1%.31,32Clinical complementary application of acupuncture and moxibustion can help control CD symptoms and complicationsin the short-term,andalso reduces the risk of generating the side effects produced by western medicines.8,33With the added use of moxibustion in CD patients,the dosage of western drugs can be gradually lessened,and quality of life can also be significantly improved.7In our study,we chose mesalamine as a positive reference for HPM in treating CD to enable comparison of the therapeutic effects and action mechanisms of the two treatments.Our results showed that HPM and mesalamine both alleviated colonic damage and suppressed systemic inflammation in CD rats,which means that HPM as a non-drug external therapy could produce similar therapeutic effects on colonic damage recovery and anti-inflammation as mesalamine for CD.Furthermore,HPM also contributes to improving the intestinal function of CD patients,and reduces the risk of gastrointestinal influences and side effects caused by oral mesalamine.9,34

Interventions

13 Beil WJ,McEuen AR,Schulz M,et al.Selective alterations in mast cell subsets and eosinophil infiltration in two complementary types of intestinal inflammation:ascariasis and Crohn's disease.Pathobiology 2002;70(6):303-313.

Figure 1 Illustration of HPM treatment

HPM was performed by successively placing herbal cakes and moxa cones at Qihai(CV 6)and bilateralTianshu(ST 25).Tianshu(ST 25)acupoints are located 5 mm lateral to the intersection of the upper 2/3 and lower 1/3 between the xiphoid and pubic symphysis,10 mm below the Qihai(CV 6)acupoint.Herbal cakes were 0.8 cm in diameter and 0.4 cm in height,mainly composed of the Chinese herbal medicine Fuzi(Radix Aconiti Lateralis Preparata).Moxa cones were made of 90 mg of well-prepared moxa in a cone that was 0.6 cm diameter at the bottom and 0.6 cm in height.HPM:herb-partitioned moxibustion.

In the MES group,mesalamine solution was administered intragastrically.Mesalamine entericcoated tablets from Kuihua Pharmaceutical Company(Heilongjiang,China)were levigated and made into a solution with distilled water.The daily mesalamine dosage was determined by converting the daily dosage foran adult male weighing 70 kg(4 g/d)to that appropriate for a rat weighing 200 g;hence,the human∶rat dosage ratio was 56∶1.Intragastric mesalamine administration was performed twice a day for 7 d.

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Rats in the NC and MC groups did not receive any intervention,but were treated in the same manner as the rats in the intervention groups in terms of grasping and fixing.

Observation of colonic lesions

The colon was separated from the symphysis pubis to the cecum,and then dissected longitudinally along the mesentery.After being washed with 0.9%physiological saline,the anatomical lesions of each colon were assessed and scored according to the Score Criteria of Colonic General Damage using a stereoscopic microscope from Olympus(Tokyo,Japan).18Next,each colon was fixed in 10%neutral-buffered formalin,and embedded in paraffin.Histological damage and inflammation of the colon in all groups were observed using an optical microscope from Olympus(Tokyo,Japan)after hematoxylin-eosin staining from Nanjingjiancheng Biotechnology(Nanjing,China).The histological scores were assessed according to the Score Criteria of Colonic Histological Damage.21

微信公众号推文的基本载体形式包括文字、图片、音频、视频、动画和链接等。从研究样本的统计数据来看，图文混合是优质推文的最主要形式，其中多图+文字的形式最多且图片均细致精美，有97篇；单图+文字形式为其次，有18篇；包含视频的推文数量较少，有10篇，无纯文字、纯图片或纯视频形式推文。由此可见，优质文章善用图文结合形式发布内容，为读者带来更直观的视觉享受，增强内容的丰富度和感染力。由于缺乏语音、视频和链接等形式，民主党派微信公众号尚未充分融合多种传播形式而体现出较强的功能。

Enzyme-linked immunosorbent assay(ELISA)

The serum concentrations of tumor necrosis factor α(TNF-α)and interleukin 1β (IL-1β)were tested using ELISA kits from Thermo Fisher Scientific(Waltham,MA,USA).ELISA procedures were performed according to the manufacturer's instructions.All samples were diluted appropriately and measured in triplicate.Each sample was successively incubated with primary antibody and secondary antibody for 30 min at 37℃.After being washed three times,a mixed solution of chromogenic agent A and B was added to each hole and incubated for 15 min away from light.The optical density value was tested at a wavelength of 450 nm using a Synergy H4 microplate reader from Bio-Tek(Winooski,VT,USA).The optical density values were obtained,and the serum concentrations of TNF-α and IL-1β were calculated based on the standard curve.

Real-time quantitative polymerase chain reaction(RT-PCR)

17 Liu G,Friggeri A,Yang Y,Park YJ,Tsuruta Y,Abraham E.miR-147,a microRNA that is induced upon Toll-like receptor stimulation,regulates murine macrophage inflammatory responses.Proc Natl Acad Sci USA 2009;106(37):15819-15824.

Western blotting

Expressions in the colon of TLR4,phosphorylated NF-κB inhibitor α (p-IκBα),phosphorylated IκB kinase α/β (p-IKKα/β),and phosphorylated NF-κB p65 proteins were measured by Western blotting.The total proteins were extracted with radio immunoprecipitation assay(RIPA)lysis buffer,and the protein concentration was tested using abicinchoninic acid kit from Beyotime Biotechnology(Shanghai,China).In each sample,40 μg of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and transferred onto a nitrocellulose membrane that was subsequently blocked with tris-buffered saline buffer containing 5%(w/v)bovine serum albumin and 0.05%(v/v)Tween-20 for 2 h,and then incubated with the primary antibody(TLR4,1∶1000;p-IKKα/β,1∶1000;p-IκBα,1∶1000;p-NF-κB p65,1∶1000)from Cell Signaling(Danvers,MA,USA)overnight at 4℃.After washing three times,the membrane was incubated with the secondary antibody(HRP+IgG,1∶10 000)from Minrui Biotechnology(Shanghai,China)for 1 h at room temperature.The protein bands on the membrane were visualized using luminol reagent solution,and detected using ChemiDoc™Imaging Systems from Bio-Rad(Hercules,CA,USA).The relative expression of proteins was calculated by referring to the β-actin protein.

Immunohistochemistry

Expressions of the total amounts of IKKα/β,IκBα,and NF-κB p65 and their locations in the colon were observed by immunohistochemistry.Colonic slides were deparaffinized and hydrated,followed by antigen retrieval in citrate buffer and endogenous peroxidase elimination in 1%hydrogen peroxide.Colonic slides were then blocked in 5%goat serum,and incubated with diluted primary antibodies(IKKα/β,1∶ 50;IκBα,1∶40;NF-κB p65,1∶80)from Santa Cruz(Dallas,Texas,USA)at 4℃overnight,and with secondary antibody from Dako-Agilent(Beijing,China)at 37℃for 30 min the next day.Diaminobenzidine(DAB)reagent was applied to visualize the target proteins on the slides,which were counterstained with hematoxylin and sealed with neutral gum;positive protein was brown and nuclei were blue under light microscopy.The integrated optical density of target proteins was measured using a BX53 microscope from Olympus(Tokyo,Japan),and the mean values of three fields on each slide were used for statistical analysis.

Statistical analysis

35 Kim H,Zhao Q,Zheng H,Li X,Zhang T,Ma X.A novel crosstalk between TLR4-and NOD2-mediated signaling in the regulation of intestinal inflammation.Sci Rep 2015;5:12018.

RESULTS

Morphological and histological analyses of colonic damage

Compared with the NC group,the morphological and histological colonic damage scores in the MC,MES,and HPM groups were significantly increased(allP＜0.001,Figure 2).Compared with the MC group,the morphological colonic damage score was decreased in the HPM group(P＜0.001,Figure 2),while the histological colonic damage scores were also decreased in the MES and HPM groups(bothP＜0.001,Figure 2).

Figure 2 Macroscopic and histopathological scores of colonic damage

A:macroscopic scores in each group.B:histopathological scores in each group.NC:normal control group,rats without any intervention;MC:model control group,rats only with model establishment;MES:mesalamine group,rats with intragastric administration,0.07 g a day per 200 g rats,twice a day,all 7 d;HPM:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.Data are presented as meridian(min-max),n=10 rats per group.aP＜0.01,compared with NC;bP＜0.01,compared with MC.

Histomorphological

Typical pathological changes seen in CD were observed in the MC group,such as damage of the partial mucosal epithelial cells and fissured ulcers(Figure 3).The connective tissue under the submucosa and the mesenchyme of the laminae propria were infiltrated by a large number of lymphocytes,and an abnormally large number of macrophages were present.There was also obvious injury of the goblet cells,and the glandular cavities were disordered.After HPM treatment,the colonic damage was improved,manifested by an integrated structure,successive mucosal epithelial cells,a large number of goblet cells under proliferation,and glandular cavities in normal order.Analogous regulatory effects were found in the MES group.

Serum concentrations of TNF-αand IL-1β

Compared with the NC group,the serum concentrations of TNF-α and IL-1β were significantly increased in the MC group(P＜0.001,Figure 4).Compared with the MC group,the serum concentrations of TNF-α and IL-1β were significantly reduced in the HPM and MES groups(allP＜0.001,Figure 4).There were no significant differences between the MES and HPM groups(P＞0.05).

Figure 3 Histomorphological changes in the rat colons(HE staining,×200)

A:normal control group,rats without any intervention;B:model control group,rats only with model establishment;C:mesalamine group,rats with intragastric administration,0.07 g a day per 200 g rats,twice a day,all 7 d;D:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.There are two pictures shown from the model control group(B),respectively indicating granuloma and a typical ulcer.HE:hematoxylin-eosin staining.

Figure 4 Expressions ofTNF-α and IL-1β in serum

A:TNF-α;B:IL-1β.NC:normal control group,rats without any intervention;MC:model control group,rats only with model establishment;MES:mesalamine group,rats with intragastric administration,0.07 g a day per 200 g rats,twice a day,all 7 d;HPM:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.Data are presented as mean±standard error of mean,n=10 rats per group for ELISA.aP＜0.001,compared with NC;bP＜0.001,compared with MC.TNF-α:tumor necrosis factor α;IL-1β:interleukin 1β;ELISA:Enzyme-linked immunosorbent assay.

Expressions of TLR4 mRNA and TLR4 protein in the colon

Compared with the NC group,the expressions of TLR4 mRNA and protein in the colon were significantly higher in the MC group(P＜0.001 andP＜0.01,respectively,Figure 5).Compared with the MC group,the expressions of TLR4 mRNA and protein in the colon were significantly lower in the HPM group(P＜0.05 andP＜0.01,respectively).The expression of TLR4 mRNA was significantly decreased in the MES group compared with the MC group(P＜0.01).

Expressions of total and phosphorylated IKKα/βin the colon

The expressions of total and phosphorylated IKKα/β in the colons of the CD rats were significantly increased compared with the NC group(P＜0.001 andP＜0.05,respectively,Figure 6).Compared with the MC group,the expressions of total and phosphorylated IKKα/β were significantly decreased in the HPM and MES groups(P＜0.001,P＜0.01,orP＜0.05).There was no significant difference between the MES and HPM groups(P＞0.05).

Figure 5 Expressions ofTLR4 mRNA and protein in the colon A:expression of TLR4 mRNA in each group;B:typical strips of TLR4 and β-actin in Western blotting;C:relative expression of TLR4 protein in each group.NC:normal control group,rats without any intervention;MC:model control group,rats only with model establishment;MES:mesalamine group,rats with intragastric administration,0.07 g a day per 200 g rats,twice a day,all 7 d;HPM:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.Data are presented as mean±standard error of mean,n=10 rats per group for PCR;n=8 rats per group for Western blotting.aP＜0.001,compared with NC;bP＜0.01,compared with NC;cP＜0.01,compared with MC;dP＜0.05,compared with MC.TLR4:Toll-like receptor 4.

Expressions of total and phosphorylated IκB-αin the colon

The total amount of IκB-α in the colon of CD rats was significantly downregulated compared with the NC group(P＜0.05,Figure 7).Compared with the MC group,the expression of total IκB-α was significantly upregulated in the HPM and MES groups(P＜0.001 andP＜0.01,respectively).There was no significant difference between the MES and HPM groups(P ＞0.05).The expression of phosphorylated IκB-α was similar in all groups(allP＞0.05).

Expressions of NF-κB p65 mRNA and phosphorylated NF-κB p65 in the colon

Figure 6 Expression of IKKα/β in the colon

A:expressions of total and phosphorylated IKKα/β in the colon.A1:integrated optical density of IKKα/β in each group;A2:typical strips of phosphorylated(p)IKKα/β and β-actin in Western blotting;A3:relative expression of p IKKα/β protein in each group.NC:normal control group,rats without any intervention;MC:model control group,rats only with model establishment;MES:mesalamine group,rats with intragastric administration,0.07 g a day per 200 g rats,twice a day,all 7 d;HPM:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.Data are presented as mean±standard deviation of mean,n=10 per group for immunohistochemistry;n=8 per group for Western blotting.aP＜0.001 compared with NC;bP＜0.05,compared with NC;cP＜0.001,compared with MC;eP＜0.01,compared with MC;dP＜0.05,compared with MC.B:positive expression of IKKα/β protein in the colon.IKKα/β was positively expressed in the cytoplasm of the colonic mucosa.B1:normal control group;B2:model control group;B3:mesalamine group;B4:herb-partitioned moxibustion group.Immunohistochemistry,magnification:×400.

Compared with the NC group,the expressions of NF-κB p65 mRNA and phosphorylated NF-κB p65 protein were significantly increased in the colons of CD model rats(P＜0.001 andP＜0.01,respectively,Figure 8).Compared with the MC group,the expressions of NF-κB p65 mRNA and phosphorylated NF-κB p65 protein were significantly decreased in the HPM and MES groups(P＜0.001 orP＜0.01).There were no significant differences between the MES and HPM groups(allP＞0.05).

DISCUSSION

In the present study,CD model rats displayed severe colonic damage such as intestinal ulcers and inflamma-tion,accompanied by diarrhea and hematochezia;furthermore,the serum concentrations of TNF-α and IL-1β were increased.HPM significantly improved colonic injury and inhibited inflammationin CD rats,as represented by mucosal healing,partial relief of inflammation,and reduction in the serum concentrations of TNF-α and IL-1β.Our study also found that the total amounts of key proteins in the TLR4/NF-κB signaling pathway(including TLR4,IKKα/β,IκBα,and NF-κB p65),and/or their phosphorylated proteins,were obviously changed in the colons of CD rats.HPM downregulated the expressions of TLR4,IKKα/β,and NF-κB p65,and upregulated the expression of the NF-κB p65 inhibitor IκBα in the colon.Our previous studies showed that HPM adjusted the expressions of miRNA-147/miRNA-205 and promoted colonic reparation in CD rats,16and it has been demonstrated that the TLR4/NF-κB signaling pathway is targeted by miRNA-147.17The present study has further confirmed that reduction of overexpressed inflammatory factors through regulation of the TLR4/NF-κB signaling pathway in the colon is the likely action mechanism by which HPM promotes colonic recovery and plays an anti-inflammatory effect in CD.

1930年，闻一多考据成果涌现，其研究方法虽与“预备时代”不同，然而此时期之研究对象和若干观念在《杜甫》中已显端倪。同年，闻一多在《国立武汉大学文哲季刊》第1卷第1期至第4期连续发表《少陵先生年谱会笺》，季镇淮称“这是先生专攻中国文学最初的成绩”［5］488。同年10月，闻一多在青岛，臧克家回忆说:

Figure 7 Expression of IκB-α in the colon

A:expressions of total and phosphorylated IκB-α in the colon.A1:integrated optical density of IκB-α in each group.A2:typical strips of phosphorylated(p)IκB-α and β-actin in Western blotting.A3:relative expression of p IκB-α protein in each group.NC:normal control group,rats without any intervention;MC:model control group,rats only with model establishment;MES:mesalamine group,rats with intragastric administration,0.07 g a day per 200 g rats,twice a day,all 7 d;HPM:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.Dcones every ,n=10 rats per group for immunohistochemistry;n=8 rats per group for Western blotting.aP＜0.05,compared with NC;bP＜0.01,compared with MC;cP＜0.001,compared with MC.B:positive expression of IκB-α protein in the colon.IκB-α was positively expressed in the cytoplasm of the colonic mucosa.B1:normal control group;B2:model control group;B3:mesalamine group;B4:herb-partitioned moxibustion group.Immunohistochemistry,magnification:×400.

Figure 8 Expression of NF-κB p65 protein in the colon

A:expressions of NF-κB p65 mRNA and protein in the colon.A1:expression of NF-κB p65 mRNA in each group;A2:typical strips of phosphorylated(p)NF-κB p65 and β-actin in Western blotting;A3:relative expression of p NF-κB p65 protein in each group.NC:normal control group,rats without any intervention;MC:model control group,rats only with model establishment;MES:mesalamine group,rats with intragastric administration,0.07g a day per 200g rats,twice a day,all 7 d;HPM:herb-partitioned moxibustion group,rats with herb-partitioned moxibustion treatment,two moxa cones every time,once a day,all 7 d.Data are presented as mean±standard deviation of mean,n=10 rats per group for PCR;n=8 rats per group for Western blotting.aP＜P＜0.001,compared with NC;bP＜0.01,compared with NC;cP＜0.05,compared with NC;dP＜0.001,compared with MC;eP＜0.01,compared with MC.B:positive location of NF-κB p65 protein in the colon.B1:normal control group;B2:model control group;B3:mesalamine medication group;B4:herb-partitioned moxibustion group.Immunohistochemistry,magnification: × 400.NF-κB p65 was positively expressed in the cytoplasm.

Before CD modeling,the rats were fasted for 24 h.According to the Morris method,the CD model was prepared by administering a mixture of TNBS and alcohol via enema.18,19The rats were anesthetized by intraperitoneal injection of 1%(w/v)pentobarbital sodium solution at 30 mg/kg.Dry ethanol and distilled water were mixed to create a 50%(v/v)alcohol solution.The TNBS enema solution was made by mixing 5%(w/v)TNBS from Sigma-Aldrich(St.Louis,MO,USA)with 50%alcohol from Sinopharm Group(Beijing,China)in a 2∶1 ratio.During the enema process,each rat was positioned with its head down and tail up.A needle filled with the TNBS solution was slowly and gently inserted6-8cmintotheanus,andthesolutionwastheninjected into the intestines.The enema dosage was 3 mL/kg.After the needle was removed,the rats were kept upside down for 1 min to prevent the solution from leaking.The enema was repeated once every 7 d for a total of four times.After modeling,two rats were randomly selected from each group to verify the success of the CD model creation.

从表1中的结果来看，经典组有83.45%的识别率，说明PNN神经网络的预测功能用于方块苗文的识别是可行的。从其他分组的结果来看，130×130的样本图像大小下，实验了130像素的所有公约数分组，网格从密集到稀疏，识别率逐渐升高，10像素组达到最优，之后识别率又下降到最低，从表中看出表现最好的10像素组的识别率比经典组的识别率高4.85%，具有很可观的提升幅度，说明此种像素分类优化方案是可行的。图10中与其他两种神经网络的横向对比来看，PNN神经网络本身的识别效果是优于BP神经网络和LVQ神经网络的，通过此种优化方案，挖掘出PNN神经网络更多的识别分类潜力。

As there are some definite and reliable therapeutic effects of HPM for CD,it is important to investigate the action mechanisms of HPM to promote its clinical application.Therefore,our study further explored the mechanisms of HPM in alleviating colonic damage and producing anti-inflammation through its effects on the TLR4/NF-κB signaling pathway.TLR4,one of the important pattern recognition receptors,is mainly activated by lipopolysaccharide,and plays a key role in the cascade activation of NF-κB signaling transduction.IκBα binds to NF-κB p65 and produces negative regulation.IKKα/β,an intracellular factor downstream of TLR4,activates and degrades IκBα to release NF-κB p65.NF-κB p65 ultimately combines with target genes in the nucleus,and regulates the transcription of inflammatory factors.35,36Moxibustion can reportedly inhibit the expression of IL-8 and promote IL-10 in the colons of rats with ulcerative colitis by influencing the TLR9/NF-κB pathway.37,38Our study focused on TLR4 as a trigger inducing the activation of the NF-κB pathway.TLR4 and TLR9 are key receptors that affect the activation of NF-κB signaling transduction.39TLR4 is positioned in the cell membrane,and is mainly expressed in various lymphocytes in the colonic mucosa;in contrast,TLR9 is intracellular,and is involved in the recognition of CpG DNA from bacteria andviruses.40,41TLR4 is stimulated earlier than TLR9 by the bacterial surface antigens,and producesthe immune defense and damage effects.It has been confirmed that HPM has a significantly regulatory effect on the TLR4/NF-κB pathway.42Our study found that HPM effectively facilitated the normalization of the expressions of TLR4,IKKα/β,IκBα,and NF-κB p65 mRNAs and/or proteins in the colons of CD rats.Our results are consistent with previous studies,verifying that regulation of TLR4/NF-κB signaling transduction is an important mechanism of moxibustion in alleviating inflammation and inhibiting the expressions of inflammatory factors.43Moxibustion can also influence the extracellular signal-regulated kinases signaling pathway in the colon fibroblasts of CD rats.19Our study and previous studies have provided comprehensive and reliable evidence that contributes to promoting the clinical application of HPM as an effective,stable,and safe therapeutic method for CD.

The present study had some limitations.Although we proved that the regulatory effects of HPM on the TLR4/NF-κB signaling pathway play an important role in the improvement of colonic injury and inhibition of colonic inflammation in CD,future research in which essential blockers are applied is needed to further confirm the hypothesized anti-inflammatory action mechanism of HPM.

种植橡胶树也会耗费大量的水，尽管种植橡胶需要大量的水，但是由于该地充沛的降水和湿热的气候，所以具备种植橡胶的自然条件。

In conclusion,HPM alleviates colonic damage and inflammation in CD rats,which is possibly achieved by the regulation of the TLR4/NF-κB signaling pathway.These findings may provide scientific evidence for the development of safe and inexpensive non-pharmacological therapies for CD in the future.

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2 Ng WK,Wong SH,Ng SC.Changing epidemiological trends of inflammatory bowel disease in Asia.Intest Res 2016;14(2):111-119.

3 Zhao J,Ng SC,Lei Y,et al.First prospective,population based inflammatory bowel disease incidence study in mainland of China:the emergence of"Western disease".Inflamm Bowel Dis 2013;9:1839-1845.

物理课程的教学实际上就是为了学生核心素养理念培养所服务的。在新课标下，在物理课程教学的过程中，教师要时刻注意不能忽视学生的实际课堂表现，一定要对他们的状态进行监督，从而及时对自己的授课方式进行修正与改进。当然在物理学科的教学中，不同教师肯定会有自己的方法或者经验，本文主要是依据笔者近些年高中物理教学过程中的实际经验总结而成的。笔者希望我们的高中物理教学课堂可以在前期的有效备课中让物理学科核心素养的培育有机融入课堂，从而让学生可以在一节节有趣的课堂中慢慢培养自身对这一学科的兴趣和爱好，最终爱上物理、爱上学习。

4 Romberg-Camps MJ,Bol Y,Dagnelie PC,et al.Fatigue and health-related quality of life in inflammatory bowel disease:results from a population-based study in the Netherlands:the IBD South Limburg Cohort.Inflamm Bowel Dis 2010;16:2137-2147.

5 Bao CH,Zhao JM,Liu HR,et al.Randomized controlled trial:moxibustion and acupuncture for the treatment of Crohn's disease.World J Gastroenterol 2014;20(31):11000-11011.

6 Langhorst J,Wulfert H,Lauche R,et al.Systematic review of complementary and alternative medicine treatments in inflammatory bowel diseases.J Crohns Colitis 2015;9(1):86-106.

7 Bao C,Liu P,Liu H,et al.Different brain responses to electro-acupuncture and moxibustion treatment in patients with Crohn's disease.Sci Rep 2016;6:36636.

那天晚上，温衡住在陶小西家的客房里，许久都睡不着，她跑去陶小西的房间，看着他沉睡的脸，心里生出一丝难过，也许她跟陶小西再也回不到小时候了。

8 Ji J,Lu Y,Liu H,et al.Acupuncture and moxibustion for inflammatory bowel diseases:a systematic review and meta-analysis of randomized controlled trials.Evid Based Complement Alternat Med 2013;2013:158352.

9 Joos S,Brinkhaus B,Maluche C,et al.Acupuncture and moxibustion in the treatment of active Crohn's disease:a randomized controlled study.Digestion 2004;3:131-139.

10 Warren BF.Classic pathology of ulcerative and Crohn's colitis.J Clin Gastroenterol 2004;38(5 Suppl 1):S33-S35.

广西多年来深入实施珠防林、海防林、退耕还林、石漠化综合治理、“绿满八桂”等造林绿化工程、公益林保护及生态环境保护建设工程，生态保护与修复稳步推进。从《2017年广西壮族自治区环境状况公报》可知：广西植被生态质量和植被生态改善程度均居全国首位；森林面积达1480万公顷，居全国第六位；森林覆盖率为62.31%，居全国第三位，西部地区第一位；石漠化综合治理工程成效突出，据第三次石漠化监测成果显示：广西石漠化土地面积减少率是8个石漠化省 （区）中最高的。

从养殖收入看，衢州市龙游县周公畈水库比重最高，达90.91%，安吉县大河口水库比重达到47.06%。这些水库的主要功能是灌溉，但由于农业灌溉用水收费已经取消，造成这些水库收入主要来源于养殖。这种情况将严重影响水库的可持续利用。

11 Hommes DW,van Deventer SJ.Anti-and proinflammatory cytokines in the pathogenesis of tissue damage in Crohn's disease.Curr Opin Clin Nutr Metab Care 2000;3(3):191-195.

12 Siegmund B,Zeitz M.Innate and adaptive immunity in IBD.World J Gastroenterol 2011;27:3178-3183.

In the HPM group,HPM was administered at bilateral Tianshu(ST 25,at the same level as the umbilicus and lateral to the anterior midline)and Qihai(CV 6,located on the midline 1.5 cun below the umbilicus).20The herb cake was especially made for the rats'size;the major constituent was Fuzi(Radix Aconiti Lateralis Pre-parata)from Shanghai Research Institute of Acupuncture and Meridian(Shanghai,China),which was levigated and mixed with yellow rice wine before being made into cakes using a mold.Two moxa cones were ignited successively at each acupoint during every HPM session,and HPM was performed once a day for 7 d(Figure 1).

14 Fukata M,Arditi M.The role of pattern recognition receptors in intestinal inflammation.Mucosal Immunol 2013;3:451-463.

15 Leal RF,Milanski M,Ayrizono Mde L,et al.Toll-like receptor 4,F4/80 and pro-inflammatory cytokines in intestinal and mesenteric fat tissue of Crohn's disease.Int J Clin Exp Med 2013;2:98-104.

16 Wei K,Zhang D,Hong J,et al.Herb-partitioned moxibustion and the miRNAs related to Crohn's disease:a study based on rat models.Evid Based Complement Alternat Med 2015;2015:265238.

Expressions of TLR4 and NF-κB p65 mRNA in the colon were detected by RT-PCR.Total RNA in the colon was extracted and amplified using a PCR kit according to the manufacturer's protocol of Thermo Fisher Scientific(Waltham,MA,USA).The primers used were:rat TLR4,upstream-primer(5′-TTCAAGGGCTGAGAGCGAGT-3′),downstream-primer(5′-CGAGGTCATTTCTGTATGCT-3′);rat NF-κB p65,upstream-primer(5'-GTTCACAGACCTGGCATCCGT-3'),downstream-primer(5'-GAGAAGTCCATGTCCGCAATG-3').RT-PCR was carried out as follows:initial denaturation at 48℃for 30 min,followed by 95℃for 15 min,40 cycles of 95℃for 15 s and 55℃for 1 min,and a final unlimited 4℃hold.The expression of target genes was measured in triplicate,and was normalized to β-actin as an internal control.Gene relative expression was calculated using the 2－ΔΔctmethod.

18 Morris GP,Beck PL,Herridge MS,Depew WT,Szewczuk MR,Wallace JL.Hapten-induced model of chronic inflammation and ulceration in the rat colon.Gastroenterology 1989;96:795-803.

三维模型的长度为105 m，高度为40 m，厚度为40 m，坡率为1∶1，边坡土质为完全固结土，数值模型单元数为76 600个。

19 Wang X,Lu Y,Wu L,et al.Moxibustion inhibits the ERK signaling pathway and intestinal fibrosis in rats with Crohn's Disease.Evid Based Complement Alternat Med 2013;2013:198282.

20 Li ZR.Experimental Acupuncture Science.2nd ed.Beijing:China Press of Traditional Chinese Medicine,2007:327-329.

21 Butzner JD,Parmar R,Bell CJ,Dalal V.Butyrateenema therapy stimulates mucosal repair in experimental colitis in the rat.Gut 1996;38(4):568-573.

22 Moja L,Danese S,Fiorino G,Del Giovane C,Bonovas S.Systematic review with network meta-analysis:comparative efficacy and safety of budesonide and mesalazine(mesalamine)for Crohn's disease.Aliment Pharmacol Ther 2015;41(11):1055-1065.

23 Criscuoli V,Modesto I,Orlando A,Cottone M.Mesalazine for the treatment of inflammatory bowel disease.Expert Opin Pharmacother 2013;14(12):1669-1678.

24 Coward S,Kuenzig ME,Hazlewood G,et al.Comparative effectiveness of mesalamine,sulfasalazine,corticosteroids,and budesonide for the induction of remission in Crohn's disease:a bayesian network Meta-analysis.Inflamm Bowel Dis 2017;23(3):461-472.

25 Iacucci M,de Silva S,Ghosh S.Mesalazine in inflammatory bowel disease:a trendy topic once again?Can J Gastroenterol 2010;24(2):127-133.

我以为，所谓教学，就是泾渭分明的教师教和学生学。于是我卖力地教，想方设法地教，结果是，我累出了咽炎和声带小结，常年吃药；学生也并没有学到太多，学到的也多是死知识，缺乏创新性。

26 Dahl JU,Gray MJ,Bazopoulou D,et al.The anti-inflammatory drug mesalamine targets bacterial polyphosphate accumulation.Nat Microbiol 2017;2:16267.

27 Bantel H,Berg C,Vieth M,Stolte M,Kruis W,Schulze-Osthoff K.Mesalazine inhibits activation of transcription factor NF-kappaB in inflamed mucosa of patients with ulcerative colitis.Am J Gastroenterol 2000;95(12):3452-3457.

28 Suzuki Y,Iida M,Ito H,Tachikawa N,Hibi T.Investigation of a high-dose pH-dependent-release mesalazine on the induction of remission in active Crohn's disease.Drugs R D 2016;16(1):35-43.

29 Pithadia AB,Jain S.Treatment of inflammatory bowel disease(IBD).Pharmacol Rep 2011;63:629-649.

30 Travis SP,Stange EF,Lemann M,et al.European evidence based consensus on the diagnosis and management of Crohn's disease:current management.Gut 2006;55(S1):i16-i35.

31 Gisbert JP,Chaparro M,Gomollón F.Common misconceptions about 5-aminosalicylates and thiopurines in inflammatory bowel disease.World J Gastroenterol 2011;17(30):3467-3478.

32 Zheng JJ.Advances and application of ammonium salicylic acid(Salt)in treating inflammatory bowel disease.Zhong Guo Chu Fang Yao 2012;10(5):8-12.

33 Opheim R,Hoivik ML,Solberg IC,Moum B,IBSEN Study Group.Complementary and alternative medicine in patients with inflammatory bowel disease:the results of a population-based inception cohort study(IBSEN).J Crohns Colitis 2012;6(3):345-353.

34 Shang HX,Wang AQ,Bao CH,et al.Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients.World J Gastroenterol 2015;21(16):4986-4996.

1.2.1 细菌培养及RNA提取 挑取单克隆细菌于LB培养基中，37℃、250 rpm培养过夜，按1∶100转接于20 mL LB培养基中，相同条件培养至特定状态，需要时加入100 μg/mL氨卞青霉素，加入0.2% L-阿拉伯糖诱导质粒目的基因的表达.快速离心收集细菌，总RNA的提取采用TRIzol法，具体操作按说明书进行，加入DNase I消化基因组DNA，核酸检测仪测定浓度，-80℃保存备用.

SPSS Statistics for Windows,version 18.0(released in 2009;SPSS Inc.,Chicago,IL,USA)was used for data analysis.The data were expressed as mean±standard error of mean(SEM)when normally distributed with homogeneity of variance.The data were expressed as median(minimum-maximum)when not normally distributed,and were displayed using a box diagram.For data with homogeneity of variance,one-way analysis of variance was performed,and the least-significant difference test was used for comparisons between two groups.For data with heterogeneity of variance,the Kruskal-WallisHtest was applied,and the Nemenyi test was used for comparisons between two groups.APvalue of＜0.05 was taken to indicate statistical significance.

36 Singh JC,Cruickshank SM,Newton DJ,et al.Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis.Am J Physiol Gastrointest Liver Physiol 2005;288(3):G514-G524.

37 Ma TM,Han Y,Ma XD,Zeng XX,Ge W.Influence of moxibustion with different duration on colonic epithelial structure,serum inflammatory cytokines,and intestinal mucosa inflammatory cell signal transduction pathways.Zhen Ci Yan Jiu 2014;39(1):20-26.

38 Han Y,Ma TM,Lu ML,Ren L,Ma XD,Bai ZH.Role of moxibustion in inflammatory responses during treatment of rat ulcerative colitis.World J Gastroenterol 2014;20(32):11297-11304.

Crohn's disease(CD)has become prevalent in recent decades.The incidence of CD is highest in western countries,with an annual rate of 29.3 per 100 000 persons.1A large-scale population-based study of eight Asian countries showed obvious variations in the incidence of inflammatory bowel disease(IBD),ranging from 0.54 to 3.44 per 100 000 persons.2The highest incidence of IBD was found in East Asian urbanized areas(Guangzhou,China)at 3.44 per 100 000 persons,with a CD incidence of 1.22 per 100 000 persons.3CD is a recognized refractory disease that causes chronic non-specific colonic inflammation.Currently,the two major therapies for CD are anti-inflammation and immunosuppression.These CD treatments have variable clinical effects;however,the efficacy and dosage of these therapies are generally proportional,which is the main reason for high dosage-induced adverse events,and has challenged their extensive and proper application.Long-term medical experience,repeated symptoms,and disorders of intestinal function have undoubtedly influenced the life quality of CD patients,and increased the burden on their families.4Therefore,it is essential to find an effective and safe therapy for CD by controlling the relapse of clinical symptoms,improving patient quality of life,and helping to reduce the side effects of efficacious medicines.

40 Lee J,Rachmilewitz D,Raz E.Homeostatic effects of TLR9 signaling in experimental colitis.Ann NY Acad Sci 2006;1072:351-355.

41 Fukata M,Abreu MT.TLR4 signalling in the intestine in health and disease.Biochem Soc Trans 2007;35(Pt 6):1473-1478.

42 Yoon HM,Jang KJ,Han MS,et al.Ganoderma lucidum ethanol extract inhibits the inflammatory response by suppressing the NF-κB and toll-like receptor pathways in lipopolysaccharide-stimulated BV2 microglial cells.Exp Ther Med 2013;5(3):957-963.

43 Yuan J,Hu L,Song XG,et al.Influence of moxibustion on TLR4-MyD88-NF-κB signal transduction pathway of synovial tissue in rheumatoid arthritis rats.Zhen Ci Yan Jiu 2015;40(3):199-204.