甲状腺相关眼病与Th1/Th2免疫平衡

背景 甲状腺相关眼病（GO）是自身免疫性疾病,目前其发病机制尚不清楚,近年来的研究发现Th1/Th2型免疫平衡反应的失调是导致GO的主要原因. 目的 采用脂质体包裹的人促甲状腺激素受体（hTSHR）胞外段基因重组质粒免疫同系雌性BALB/c小鼠以建立GO动物模型,研究关于在GO小鼠模型血清Th1/Th2型免疫反应平衡状态,探讨GO的病理机制.方法 采用计算机数字随机分配法将同系6-8周龄雌性BALB/c小鼠32只分为重组质粒注射组、空白对照组、空质粒注射组和脂质体注射组.重组质粒注射组分别于实验的0、3、6周采用脂质体包裹的重组质粒pcDNA3.1 ＋/hTSHR289各30 μg行双胫前肌内注射,再于腹腔内注射40 μg以免疫小鼠,空质粒注射组和脂质体注射组分别采用pcDNA3.1＋和脂质体以同样的方法进行免疫,空白对照组不做任何处理.分别于注射前及各次注射后第4天及第17周末处死小鼠行心脏采血,采用ELISA法检查各组小鼠血清中白细胞介素（IL）-2及IL-4质量浓度,计算IL-2/IL-4值.于初次免疫后17周末处死重组质粒注射组小鼠,取甲状腺及眼眶组织制作病理切片,采用苏木精-伊红染色法检测标本组织中炎性细胞浸润情况,对有炎性细胞浸润的标本进行免疫组织化学检测,计算B细胞特异标志物CD20阳性细胞（B）与T细胞特异标志物CD3ε阳性细胞（T）的比值,B∶T＞1.0者为Th2反应,相反者为Th1反应.结果 在第2次和第3次注射后,重组质粒注射组小鼠血清中IL-2质量浓度明显低于空白对照组、脂质体注射组、空质粒注射组,差异均有统计学意义（均P＜0.05）,但组间小鼠血清IL-4质量浓度差异无统计学意义（P＞0.05）;重组质粒注射组小鼠在第2次、第3次免疫后血清IL-2质量浓度明显低于免疫前及第1次免疫后,差异均有统计学意义（注射前：P=0.009、0.019;第1次注射后：P=0.002、0.004）,而处死时小鼠血清IL-2质量浓度有所回升,明显高于第3次免疫后IL-2质量浓度[（44.31±1.77） pg/ml与（42.43±2.29） pg/ml],差异有统计学意义（P=0.031）.空白对照组、空质粒注射组、脂质体注射组、重组质粒注射组第2次、第3次注射后血清IL-4质量浓度及IL4/IL2值均明显高于注射前和第1次注射后,其处死时重组质粒注射组小鼠血清IL-4质量浓度明显高于第3次注射后,差异均有统计学意义（均P＜0.05）.处死时重组质粒注射组苏木精-伊红染色有炎性细胞浸润的7只小鼠12个眼眶标本中4个眼眶B∶T值＞1.0. 结论 采用脂质体包裹的TSH受体胞外段基因重组质粒免疫同系雌性BALB/c小鼠可成功建立GO动物模型,重组质粒注射组小鼠在初次免疫后17周内以Th1型反应为主,小鼠第2次免疫后免疫平衡开始向Th2型转变.

Hydrolyzed seawater pearl tablet modulates the immunity via attenuating Th1/Th2 imbalance in an immunosuppressed mouse model

OBJECTIVE: To investigate whether Hydrolyzed Seawater Pearl tablet(HSPT) could modulate the Th1/Th2 imbalance in an immunosuppressed mouse model with Th1 to Th2 shift induced by Cyclosporine A(Cs A) which can be used in the clinical treatment of Th2 to Th1 shift diseases, and explore the possible mechanism for the adjuvant therapeutic efficacy of HSPT on recurrent respiratory infections(RRI) and acquired immune deficiency syndrome(AIDS).METHODS: The mice were randomly divided into six groups of five animals each, namely normal group, model group, lentinan polysaccharide tablet(LPT) group and three HPST treated groups. HPST treated groups were administered with HPST(0.51,1.02, 2.04 g/kg) via intragastric gavage(i.g) for 30 consecutive days. LPT used as reference drug for positive control, LPT group was administered with LPT(8.2 mg/kg) for 30 consecutive days. Normal group and model group were received distilled water. The animals in model group, LPT group and HPST treated groups were injected intraperitoneally with Cs A(50 mg/kg) to establish the immunosuppressed mice model with Th1 to Th2 shift on the20 th, 22 nd and 24 th day, one hour after the administration of the respective treatment. Animals were sacrificed one hour after the last administration to collect blood and splenic tissue. The proportion of T cells including CD8+ and CD4+ T cells, Th1 and Th2 in peripheral blood of experimental mice were measured by flow cytometric. The protein level in serum and m RNA level in splenic tissue of experimental mice for interleukin(IL)-2, IL-12, interferon-γ(IFN-γ), IL-4, IL-6, IL-10 and IL-13 were measured by enzyme linked immunosorbent assay and fluorescence quantitative polymerase chain reaction respectively.RESULTS: HSPT elevated the proportion of T cells including both CD8+ and CD4+ T cells, in which the proportion of Th1 and Th2 cells increased, while the ratio of Th1/Th2 cells decreased in peripheral blood of the immunosuppressed mouse model with Th1 to Th2 shift induced by Cs A. Furthermore,HSPT elevated both protein and m RNA level of Th1-type cytokines IL-2 and IFN-γ, while had no significant effect on protein and m RNA level of Th1-type cytokine IL-12 and Th2-type cytokines IL-4, IL-6, IL-10, IL-13 in mouse model.CONCLUSION: Our findings suggest that HSPT can increase proportion of T cells including both CD8+and CD4+T cells and induce Th2 to Th1 shift in both cells and cytokines, which probably was the mechanism to account for the adjuvant therapeutic efficacy of HSPT on RRI and AIDS.

Wang-Bi tablet, a patented Chinese medicine, maintains the balance of Th1/Th2 in mice with collagen-induced arthritis

OBJECTIVE: To investigate the pharmacological mechanism of Wang-Bi tablets(WBTs), a Chinese patented medicine, in rheumatoid arthritis(RA) using mice with collagen-induced arthritis(CIA).METHODS: A mouse model of CIA was induced using bovine type Ⅱ collagen. WBT treatment was administered and efficacy was evaluated. The levels of interferon-γ(IFN-γ), interleukin-2(IL-2), and interleukin-4(IL-4) were examined using an enzyme-linked immunosorbent assay, and the proportions of Th1 and Th2 were detected using flow cytometry. T-bet and GATA-binding protein 3(GATA3)expression were demonstrated using Western blot analysis.RESULTS: Paw swelling and the arthritis index decreased significantly following WBT treatment. Histopathological analysis revealed markedly alleviated damage to synovium tissue in the WBT and methotrexate treatment groups. WBT regulated the production of IFN-γ, IL-2, and IL-4 and modulated Th1 and Th2 cell populations, which might have been induced by the attenuation of Th1 and Th2 cell differentiation through a decrease in the expression of T-bet and an increase in the expression of GATA3 in the synovial tissue in CIA mice.CONCLUSION: These results indicate that WBT may produce a therapeutic effect on CIA through maintaining the balance of Th1/Th2 cells, which could result in a decrease in the autoinflammatory disorder observed in RA.

Effects of appendectomy and oral tolerance on dextran sulfate sodium colitis

AIM： To evaluate the concomitant effects of appendec- tomy and oral tolerance on colitis.METHODS： Delayed-type hypersensitivity （DTH） was investigated at a 7-d interval after ovalbumin （OVA） ad- ministration and immunization under normal and colitis conditions in appendectomized or sham-operated mice. Pathological scores for the colon were graded after in- gestion of colon-extracted protein （CEP） and induction of dextran sulfate sodium （DSS） colitis in appendecto- mized or sham-operated mice. Thereafter, Thl and Th2 in Peyer＇s patches and spleen lymphocytes were de- tected in CEP-treated and bovine serum albumin （BSA）-treated control mice.RESULTS： In appendectomized mice, DTH was not inhibited at day 7 after OVA administration and at the initial phase of DSS colitis, whereas it was inhibited at day 14 and day 21. However, in sham-operated mice, it was inhibited during the whole procedure and the onset of DSS colitis. The protective role of CEP against DSScolitis was present in sham-operated mice, with pre- dominant improvement of colonic pathological changes, while vanished in the appendectomized mice. A shift from Thl to Th2 in Peyer＇s patches resulted from a de- crease of Thl cells with the ingestion of CEP. Compared with BSA in the sham-operated group, no predominant changes were observed in the appendectomized mice.CONCLUSION： Appendectomy interferes with the pro- tective role of CEP in DSS colitis via a shift from Th2 to Thl during oral tolerance induction.

Protective effects of catalpol and rhein in murine experimental autoimmune encephalomyelitis via regulation of T helper (Th)1, Th2,Th17, and regulatory T cell responses

OBJECTIVE:To examine the effects of catalpol and rhein on pro-and anti-inflammatory responses in C57 BL/6 mice with experimental autoimmune encephalomyelitis(EAE),a model of multiple sclerosis.METHODS:Female C57 BL/6 mice were randomly divided into four groups(n=30):(a)normal salinecontrol,(b)EAE control,(c)EAE+prednisone acetate(PA,6 mg/kg),and(d)EAE+catalpol(40 mg/kg)and rhein(5 mg/kg).EAE was induced by injection of myelin oligodendrocyte glycoprotein 35-55 plus pertussis toxin.Treatments were orally administered daily for 40 d.Disease progression and neurological function were assessed using a semi-quantitative scale of tail and limb paralysis.Brains and spinal cords were collected on Days 6,20,and 40 and assessed for histopathological changes by hematoxylin and eosin staining.Production of interleukin(IL)-2,IL-4,IL-10,and IL-17 A protein was measured by enzyme-linked immunosorbent assay.Expression of the T helper(Th)1-,Th2-,Th17-,and regulatory T cell(Treg)-specific transcription factors T-bet,GATA3,ROR-γt,and Foxp3,respectively,were analyzed by quantitative reverse-transcription polymerase chain reaction and western blot analysis.RESULTS:Combination treatment with catalpol and rhein significantly alleviated the clinical disability and neurological dysfunction of mice with EAE.Catalpol and rhein treatment also reduced the infiltration of pro-inflammatory T cells into pathological lesions;significantly increased the expression of the anti-inflammatory factors GATA3,Foxp3,IL-4,and IL-10;and significantly decreased the expression of the pro-inflammatory factors T-bet,ROR-γt,IL-2,and IL-17 A.CONCLUSION:Catalpol and rhein reduced the neurological disabilities of mice with EAE,at least in part by rebalancing the pro-and anti-inflammatory environment in the brains and spinal cords.

Effect of chiropractic manipulation on disrupted epithelium barrier and its mechanism of specialized pro-resolving mediators in a spleen-deficiency murine model

OBJECTIVE: To investigate the influence of spleen deficiency on the epithelial barrier of jejunum and lungs in a rat model of spleen-deficiency and the effect and potential specialized pro-resolving mediators (SPMs) mechanism of chiropractic manipulation. METHODS: Three-week-old male Sprague-Dawley rats were divided randomly into normal control group (n = 6), spleen-deficiency group (n = 5) and chiropractic group (n = 6). Spleen-deficiency model was induced in spleen-deficiency group and chiropractic group. Moreover, chiropractic manipulation was performed in chiropractic group. Four weeks later, systemic Th1/Th2 balance was evaluated by the ratio of plasma interferon (IFN)-γ/interleukin (IL)-4 levels by enzyme-linked immunosorbent assay (ELISA). Epithelial barrier integrity were assessed by the observation of morphological changes by hematoxylin-eosin staining and zonula occludens (ZO)-1 gene expressions by quantitative real time polymerase chain reaction in jejunum and lungs. Plasma resolvin D1 (RvD1) and lipoxin A4 (LXA4) levels were measures by ELISA for endogenous SPMs production. The levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) in jejunum and lungs were also measured by HPLC-MS/ MS. RESULTS: Comparing with normal control group, spleen-deficiency group showed disrupted mucosa in jejunum, inflammatory condition in lungs, significantly decreased ratio of plasma IFN-γ/IL-4 levels and lower expressions of ZO-1 mRNA in both jejunum and lung tissues. Comparing with spleen-deficiency group, chiropractic group had less disrupted mucosa in jejunum and inflammatory condition in lungs, significantly increased systemic ratio of IFN-γ/IL-4 and expressions of ZO-1 mRNA in both jejunum and lung tissues. Chiropractic group had significantly enhanced plasma levels of RvD1 and LXA4, but had no significantly higher levels of DHA and AA in jejunum and lungs when comparing with spleen-deficiency group. CONCLUSION: Spleen deficiency caused systemic Th1/Th2 imbalance towards Th2 polarization and epithelial barrier disruption in jejunum and lungs.Chiropractic manipulation helped enhance endogenous SPMs production, which might be one of the action mechanism of chiropractic manipulation on the improvement of epithelial barrier disruption.

Notch signaling pathway mediates the immunomodulatory mechanism of Yangfei Huoxue decoction alleviating bleomycin-induced pulmonary fibrosis in rats

OBJECTIVE: To investigate the immunomodulatory mechanism by which Yangfei Huoxue decoction(YHD) alleviates bleomycin(BLM)-induced pulmonary fibrosis(PF) in rats.METHODS: Rats were randomly divided into two time-point groups(day 14 and 28), and each timepoint group comprised the following six subgroups: control, BLM, dexamethasone(DXM), YHD high dose(YHD-H), YHD middle dose(YHD-M), and YHD low dose(YHD-L). Haematoxylin and eosin and Masson staining, flow cytometry, enzymelinked immunosorbent assay, Western blotting and UPLC-QT of analyses were examined.RESULTS: The results showed that YHD reduced the degree of alveolar inflammation and fibrosis;downregulated the expression of CD28, CD80,CD86, Delta-like 1, Notch2, and Notch3;and upregulated the proportions of Th1/Th2 and Tc1/Tc2. The seven components of YHD were detected.CONCLUSION: The current study indicates that YHD mainly functions by regulating the immune system and that the molecular mechanism may be related to the regulation of the Notch signaling pathway.

Effect of Xinfeng capsule on pulmonary function in a adjuvant arthritis rat model

OBjECTIVE:To observe the relationship between reduced pulmonary function and regulatory T cells(Tregs)and helper T cells(Th)1/Th2 drift in a rat model of adjuvant arthritis(AA),and to study the impact of Xinfeng capsule(XFC)on pulmonary function and investigate the mechanism of action.METHODS:Forty rats were randomly divided into normal control group(NC),model control group(MC),Tripterygium glycosides tablet group(TPT),and XFC group,with 10 in each.Except for the NCgroup,AA was induced in all rats by intracutaneous injection of 0.1 mL Freund's complete adjuvant in the right paw.On the 19th day after modeling,the NC and MC groups were given physiological saline(0.9%),while the TPT and XFC groups were given TPT(10 mg/kg)and XFC(2.4 g/kg),once daily,respectively.Thirty days after administration,changes in paw swelling,arthritis index(AI),pulmonary function,levels of serumγ-interferon(IFN-γ)and interleukin(IL)-4,Tregs in peripheral blood,and IFN-γ,IL-4,Forkhead box transcription factor 3(FoxP3)in lung tissue were observed by enzyme-linked immunosorbent assay,flow cytometry,polymerase chain reaction,and western blot.RESULTS:Compared with the NC group,paw swelling,AI,IFN-γ,and Th1/Th2 were increased,and pulmonary function parameters,IL-4,FoxP3 were decreased significantly in the MC group(P<0.05 or P<0.01).Pulmonary function parameters,Treg,IL-4,FoxP3(and mRNA)were higher,and paw swelling,AI,and IFN-γ(and mRNA)were lower in the XFC group than those in the MC group.The XFC group was also much better than theTPT group in improving pulmonary function,FoxP3 mRNA,IFN-γ,IL-4,Th1/Th2,and IL-10(P<0.05 or P<0.01).CONCLUSION:Xinfeng capsule can improve pulmonary function by regulating the levels of Tregs,inhibiting the activation of Th1 to Th2 cells,inducing drift,maintaining cell immune suppression,correcting the imbalance of Th1/Th2,and reducing inflammatory mediators.

Clinical observation of point injection at Zusanli(ST36)plus abdominal point application for gastrointestinal dysfunction after laparoscopic surgery

Objective:To observe the effect of point injection at Zusanli(ST36)plus abdominal point application on gastrointestinal dysfunction after laparoscopic surgery.Methods:A total of 204 patients with gastrointestinal dysfunction after laparoscopic surgery were recruited and divided into four groups using the random number table method,with 51 cases in each group.The control group received conventional postoperative intervention.In addition to the treatment in the control group,the point injection group was given point injection at Zusanli(ST36),the application group was offered abdominal point application,and the integrated group received point injection at Zusanli(ST36)and abdominal point application.The treatment lasted 3 consecutive days in all four groups.The recovery time of gastrointestinal function indicators and the incidence rate of postoperative nausea and vomiting(PONV)were observed and recorded.Before and after treatment,the visual analog scale(VAS)was used to assess abdominal pain intensity,the venous blood type 1 helper T cells/type 2 helper T cells(Th1/Th2)was determined,the serum levels of interleukin(IL)-6 and interferon(IFN)-γwere detected using the enzyme-linked immunosorbent assay,and the plasma levels of motilin and gastrin were measured using radioimmunoassay.Results:Compared to the control group,the first exhaust time,the first defecation time,and the time of restoring fluid diet came earlier in the other three groups(P<0.05)and were earlier in the integrated group than in the point injection and application groups(P<0.05).The point injection,application,and integrated groups had a lower PONV incidence rate than the control group,and the integrated group was lower than the point injection and application groups(P<0.05).The intra-group comparisons showed that the VAS score and the levels of IL-6 and INF-γdecreased after treatment in all four groups(P<0.05);the point injection,application,and integrated groups were lower than the control group(P<0.05),and the integrated group was lower than the point injection and application groups(P<0.05).The intra-group comparisons also demonstrated that the levels of Th1/Th2,motilin,and gastrin increased after the intervention in the four groups(P<0.05);the point injection,application,and integrated groups were higher than the control group(P<0.05),and the integrated group was higher than the point injection and application groups(P<0.05).Conclusion:Point injection at Zusanli(ST36)plus abdominal point application can encourage postoperative exhaust,defecation,and the recovery of diet fluid,alleviate postoperative abdominal pain,reduce PONV,balance Th1/Th2,and regulate the secretion of motilin and gastrin in patients with gastrointestinal dysfunction after laparoscopic surgery.