缬沙坦对免疫性大鼠肝纤维化的影响

目的研究血管紧张素Ⅰ型(AT1)受体阻断剂对免疫性大鼠肝纤维化的影响。方法采用猪血清诱导建立肝纤维化模型,以大剂量和普通剂量缬沙坦干预,观察肝组织纤维化程度、胶原表达变化;免疫组化染色观察α平滑肌肌动蛋白(αSMA)、转化生长因子β1(TGFβ1)及核因子(NF)κBp65表达情况。结果与模型组比较,治疗组胶原面积显著减少,具有剂量依赖性(P<0.05),纤维化程度也明显改善;模型组αSMA、TGFβ1、NFκBp65表达增强(P<0.05),缬沙坦治疗后,都有不同程度的减弱,模型组和大剂量组αSMA分别为19.68±1.28和8.66±1.72,TGFβ1分别为22.36±4.77和9.95±2.28,NFκBp65分别为30.31±4.16和19.80±1.96(均为面密度值,P<0.05)。结论缬沙坦能明显抑制猪血清诱导的大鼠肝纤维化发生,可能与抑制肝星状细胞活化、增殖,降低TGFβ、NFκBp65等表达有关。

补中益气颗粒对自身免疫性甲状腺炎模型大鼠小肠肠黏膜超微结构的影响

目的通过观察补中益气颗粒对实验性自身免疫性甲状腺炎(Experimental autoimmune thyroiditis,EAT)模型大鼠小肠肠黏膜超微结构的影响,探讨其改善桥本氏甲状腺炎的作用机制。方法36只Sprague-Dawley(SD)大鼠,随机分为4组,分别为空白组、模型组、补中益气组、硒酵母组4组。除空白组外,其余组大鼠采用高碘喂养法,联合猪免疫球蛋白和弗氏佐剂混合免疫注射法建立EAT大鼠模型。造模后,补中益气组和硒酵母组大鼠分别灌胃补中益气颗粒和硒酵母片。56 d后检测各组大鼠甲状腺过氧化物酶抗体(Thyroid peroxidase antibody,TPOAb)、甲状腺球蛋白抗体(Thyro⁃globulin antibody,TGAb)、血清连蛋白(Zonulin)含量,分离大鼠甲状腺与小肠,在光镜(Light microscope,LM)下观察空白组与模型组甲状腺组织病理变化、各组大鼠小肠组织病理变化,在透射电镜(Transmission electron microscope,TEM)下观察大鼠小肠肠黏膜上皮细胞(Intestinal mucosal epithelial cells,IPC)超微结构变化。结果与空白组比较,模型组血清TPOAb、TGAb、Zonulin含量明显上调(P<0.05)。与模型组比较,补中益气组、硒酵母组血清TPOAb、TGAb、Zonulin含量明显下降(P<0.05)。与空白组比较,LM下模型组大鼠甲状腺组织中有大量淋巴细胞浸润,甲状腺滤泡萎缩、消失,上皮细胞嗜酸性变,小叶间隔增宽,间质纤维化,提示造模成功。EAT大鼠肠绒毛结构紊乱,上皮细胞脱落,固有层裸露,杯状细胞减少,炎性细胞浸润,TEM下IPC微绒毛变短而厚,细胞间隙扩大,紧密连接受损,线粒体肿胀、嵴断裂、基质变淡,细胞器超微结构明显受损。与模型组比较,补中益气组肠绒毛、细胞间紧密连接、细胞器结构受损减轻。结论补中益气汤可以减轻EAT大鼠小肠肠黏膜超微结构损害。

转化医学理念指导下的芍药甘草汤对实验性免疫性葡萄膜炎模型大鼠作用机制研究

目的 ：观察不同配比的芍药甘草汤对实验性自身免疫性葡萄膜炎（EAU）大鼠眼部体征、血清、脾脏及腹股沟淋巴结IL-17的影响,探讨芍药甘草汤干预葡萄膜炎的机制。方法：采用光感受器间维生素A类结合蛋白（IRBP）混合完全弗氏佐剂（CFA）及结核杆菌反复3次皮下注射建立EAU大鼠模型,不同配比的芍药甘草汤灌胃干预;采用数码裂隙灯动态观察免疫后各组大鼠的眼部炎症表现;ELISA法检测白细胞介素-17（IL-17）表达量的变化。结果：不同配比的芍药甘草汤均可减轻EAU大鼠眼部炎性病变,不同程度的恢复大鼠体内IL-17表达,且以3∶1组效果佳。结论：芍药甘草汤以3∶1配比可以有效减轻葡萄膜炎眼部的炎症改变,其机制可能与下调致炎因子IL-17表达有关。

祛风活血丸对EAU大鼠模型TGF-β2表达的影响

目的通过观察祛风活血丸对实验性自身免疫性葡萄膜炎(EAU)大鼠模型葡萄膜TGF-β2表达的影响,探讨祛风活血丸对EAU模型大鼠的治疗效果。方法将60只Lewis大鼠随机分为6组,分别为空白对照组、模型对照组、阳性对照组、祛风活血丸低、中及高剂量组,每组10只。空白对照组不作处理,其余均予以右后足垫注射光感受器间维生素A类结合蛋白多肽片断(IRBP),建立EAU大鼠模型,造模成功后进行灌胃。空白对照组与模型对照组予以蒸馏水灌胃,祛风活血丸低、中、高剂量组分别予以不同剂量祛风活血丸灌胃,阳性对照组予以熊胆开明片灌胃。分别于灌胃后第7、14、21、28 d分批处死大鼠,取眼球做免疫组化,检测TGF-β2的表达。结果 TGF-β2免疫组化:各时间点模型对照组与空白对照组相比,差异均有统计学意义(P<0.01);祛风活血丸各组大鼠与空白对照组相比,葡萄膜TGF-β2含量明显降低,各时间点差异均有统计学意义(P<0.01),其中以中、高剂量组最为明显;低剂量组于给药后第7及21 d差异有统计学意义(P7<0.01;P21<0.05);第14及第28 d差异无统计学意义(P>0.05);阳性对照组于给药后各时间点差异均有统计学意义(P14,21<0.01;P7,28<0.05)。结论祛风活血丸能通过调节免疫因子TGF-β2的状态,对大鼠葡萄膜炎发挥一定程度的治疗作用。

芍药甘草汤治疗葡萄膜炎的网络药理学研究与实验验证

目的:通过网络药理学预测芍药甘草汤主要活性成分治疗葡萄膜炎的作用机制,并选择T细胞受体信号通路所介导T细胞转化后的效应分子γ干扰素(IFN-γ)、白细胞介素(IL)-10进行验证。方法:检索中药系统药理学数据库与分析平台(TCMSP)、中药分子机制生物信息学分析工具库(BATMAN-TCM),筛选芍药甘草汤成分、靶点和葡萄膜炎靶点。使用Cytoscape 3.7.2软件构建成分靶点网络、成分疾病靶点网络、靶点相互作用网络。拓扑分析筛选关键靶点后使用软件插件ClueGO进行基因本体(GO)、京都基因和基因组百科全书(KEGG)分析。采用光感受器间维生素A类结合蛋白混合完全弗氏佐剂及结核杆菌皮下注射复制实验性自身免疫性葡萄膜炎大鼠模型,HE染色检测视网膜炎症变化,酶联免疫吸附试验(ELISA)检测血清IFN-γ、IL-10含量。结果:芍药甘草汤中47种候选成分(白芍35种、炙甘草12种),涉及治疗葡萄膜炎作用靶点164个,拓扑分析关键靶点33个。富集分析结果表明,芍药甘草汤治疗葡萄膜炎可能与细胞对活性氧的反应、多种细胞增殖与分化、一氧化氮与神经递质等分子的代谢调节、IL-6的细胞反应等生物过程有关,涉及IL-17信号通路、TNF信号途径、Th17细胞分化、T细胞受体信号通路、Toll样受体信号通路、PI3K-AKT信号通路等。芍药甘草汤可缓解大鼠视网膜炎症,降低炎症评分(P<0.01),减少IFN-γ和增加IL-10的含量(均P<0.01)。结论:芍药甘草汤治疗葡萄膜炎过程涉及多个重要炎症与免疫通路,这将成为研究其作用机制的重要参考。

Low immunogenicity of endothelial derivatives from rat embryonic stem cell-like cells

Embryonic stem cells （ESC） are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immunogenic by the expression of a normal set of MHC molecules. This immunogenicity might trigger rejection after application in regenerative therapies. In this study MHC expression of and immune responses to endothelial derivatives of rat embryonic stem cell-like cells （RESC） under inflammatory conditions were determined in comparison to primary rat aortic endothelial cells （ECs）. Cellular as well as humoral allo-recognition was analyzed in vitro. In addition, im- mune reactions in vivo were assessed by allo-antibody production and determination of interferon-y （IFNy）-secreting alio-reactive T cells. RESC derivatives expressed low but significant levels of MHC class I, and no MHC class II. In response to IFNy MHC class I expression was enhanced, while class II transactivator induction failed completely in these cells; MHC class II expression remained consistently absent. Functionally, the RESC derivatives showed a reduced allo-stimulatory capacity, protection against humoral allo-recognition in vitro and a slightly diminished sus- ceptibility to cytotoxic T cell lysis. Furthermore, in vivo experiments demonstrated that these ceils do not trigger host immune reactions, characterized by no allo-antibody production and no induction of allo-reactive memory T cells. Our results show that endothelial derivatives of RESC have a distinctive reduced immunogenic potency even under inflammatory conditions.

Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis.Expression of α-smooth muscle actin (α-SMA) and collagen typesⅠand Ⅲ was evaluated by real-time PCR.Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method.Finally,collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay.RESULTS:The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen.siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner.Moreover,HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs.CONCLUSION:This study suggests a significant functional role for HMGB1 in the development of liver fibrosis.It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

Expression of Angiotensin Ⅱ and Aldosterone in Radiation-induced Lung Injury

Objective Radiation-induced lung injury （RILl） is the most common, dose-limiting complication in thoracic malignancy radiotherapy. Considering its negative impact on patients and restrictions to efficacy, the mechanism of RILl was studied. Methods Wistar rats were locally irradiated with a single dose of 0, 16, and 20 Gy to the right half of the lung to establish a lung injury model. Two and six months after irradiation, the right half of the rat lung tissue was removed, and the concentrations of TGF-[31, angiotensin II, and aldosterone were determined via enzyme-linked immunosorbent assay. Results Statistical differences were observed in the expression levels of angiotensin II and aldosterone between the non-irradiation and irradiation groups. Moreover, the expression level of the angiotensin II-aldosterone system increased with increasing doses, and the difference was still observed as time progressed. Conclusions Angiotensin II-aldosterone system has an important pathophysiological function in the progression of RILI.

Effect of moxibustion at Shenque (CV8) on immune system in rats with different degrees of exhaustive exercise

Objective: To investigate the effect of moxibustion at Shenque (CV8) on the immune system in rats with different levels of exhaustive exercise. Methods: Fifty-six male Sprague-Dawley (SD) rats were randomly divided into a blank group (n=8), an exhaustive group (n=24), and a moxibustion group (n=24). The exhaustive group was randomly divided into a 1-time exhaustive group, a 4-time exhaustive group and a 7-time exhaustive group, with 8 rats in each group. According to the treatment time, the moxibustion group was randomly divided into a 1-time moxibustion group, a 4-time moxibustion group and a 7-time moxibustion group, with 8 rats in each group. Rats in the exhaustive groups and the moxibustion groups were subjected to replicating the exhaustive swimming models. Rats in each moxibustion group received mild moxibustion for 15 min immediately after the exhaustive modeling, once every other day. Twenty-four hours after the corresponding exhaustive exercise, the rats in each group were tested for the levels of serum immunoglobulin (Ig) G, IgA, IgM and acid phosphatase (ACP), and the morphological changes of spleen tissues were observed. The level of IgA was detected by immunoturbidimetric assay, and the levels of IgG, IgM and ACP were detected by en zyme-linked imm uno sorb ent assay (ELISA). Results: Compared with the 1-time exhaustive group, swimming time of rats in the 4-time exhaustive group was significantly proIonged (P<0.01), and swimming time of rats in the 7-time exhaustive group was significantly shortened (P<0.01). Compared with the 7-time exhaustive group, exhaustive swimming time of rats in the 7-time moxibustion group was significantly proIonged (P<0.01). Compared with the blank group, the IgG level in the 1-time exhaustive group was significantly decreased (PvO.Ol), and the levels of IgG, IgA and IgM in the 4-time exhaustive group and the 7-time exhaustive group were all sign ifica ntly decreased (P<0.05 or PvO.Ol), while the ACP level was in creased sign ifica ntly (both P<0.01). Microscopically, the number of splenic corpuscles in the 1-time exhaustive group was reduced;the center of some splenic corpuscles in the 4-time exhaustive group was damaged;the number of splenic corpuscles in the 7-time exhaustive group was reduced, and there was no obvious germinal center. Compared with the 4-time exhaustive group, the IgA level in the 4-time moxibusti on group was sign ifica ntly in creased (P<0.01), and the ACP level was sign ifica ntly decreased (P<0.01). Compared with the 7-time exhaustive group, the levels of IgG, IgA and IgM in the 7-time moxibustion group were sign ifica ntly in creased (all PvO.Ol), and the ACP level was sign ifica ntly decreased (P<0.01). Microscopically, the nu mber of splenic corpuscles in the 1-time moxibustion group was reduced;the center of some splenic corpuscles in the 4-time moxibustion group was damaged together with hyperplasia of some splenic corpuscles;blast cells were proliferated in the center of some splenic corpuscles in the 7-time moxibustion group. Conclusion: Moxibustion at Shenque (CV 8) can improve the levels of IgG, IgA and IgM, reduce the ACP level, repair damaged spleen tissues, and enhance the immunity of the body to some extent in the Iong-term fatigue rats.

Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model

Liver transplantation is an established therapy for end-stage liver diseases. Graft rejection occurs unless the recipient receives immunosuppression after transplantation. This study aimed to explore the mechanism of acute rejection of liver allografts in rats pre-treated with total body irradiation to eliminate passenger lymphocytes and to define the role of CD4＋CD25＋ regulatory T cells in the induction of immunotolerance in the recipient. Male Lewis rats were used as donors and male DA rats were re- cipients. Rats were randomly assigned to the following four groups： control group, homogeneity liver transplantation group, idio-immunotolerance group and acute rejection group. After transplantation, the survival time of each group, serum alanine aminotransferase, total bilirubin levels, number of Foxp3＋CD4＋CD25＋ regulatory T cells, expression of glucocorticoid-induced tumor necrosis factor receptor on T cell subgroups, histopathology of the hepatic graft and spleen cytotoxic T lymphocyte lytic activity were measured. In the acute rejection group, where donors were preconditioned with total body in＇adiation before liver transplantation, all recipients died between day 17 and day 21. On day 14, serum alanine aminotransferase increased signifi- cantly to （459.2±76.9） U L^- 1, total bilirubin increased to （124.1±33.7） μmol L-1 （P〈0.05） and the ratio of Foxp3＋CD4＋CD25＋ regulatory T cells decreased significantly to 1.50%±0.50% （P〈0.05） compared with the other groups. Analysis of the T cell subpopulations in the acute rejection group varied from the other groups. Histological analysis showed typical changes of acute rejection in the acute rejection group only. Preconditioning of the donors with total body irradiation eliminated passenger lymphocytes of the liver graft, and thus affected the course of tolerance and induced acute rejection after liver transplantation.

Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats

Elucidation of the mechanisms of liver fibrogenesis is important to treat liver fibrosis.In this study,we established rat models of liver fibrosis with stages from 0–1,2,and 3–4 to 4 at 2,4,6,and 8 weeks,respectively,by injection of pig serum.Liver fibrogenesis was detected by Masson’s trichrome staining.Rat non-parenchymal cells(NPCs)were enriched 4-fold by Percoll density gradient centrifugation.Protein extracts from NPCs were prepared at 4 and 8 weeks,separated by two-dimensional electrophoresis,and then stained with Coomassie Blue G-250.At 4 weeks,we identified 18 non-redundant differentially expressed proteins of which protein disulfide-isomerase associated protein 3(PDIA3)and NDUV showed consistent expression at protein and mRNA levels from 4 to 8 weeks.PDIA3 was found to be down-regulated by Western blotting in the rat model and immunohistochemically in human liver.Our results revealed important aspects of the pathogenesis/progression of liver fibrosis and demonstrated important changes in protein expression levels of NPCs at various stages of liver fibrosis.

Protective effect of Sijunzi decoction on neuromuscular junction ultrastructure in autoimmune myasthenia gravis rats

OBJECTⅣE: To investigate the protective role of Sijunzi decoction in neuromuscular junction(NMJ)and muscle cell mitochondria ultrastructure; as well as its effects on the amount of adenosine triphosphate(ATP) and the activities of mitochondrial respiratory chain complexes I, Ⅱ, Ⅲ, and Ⅳ in autoimmune myasthenia gravis rats.METHODS: An experimental autoimmune myasthenia gravis(EAMG) rat model was established by inoculating rats with acetylcholine receptors extracted from Torpedo. Rats were divided into three groups: model, prednisone, and Sijunzi decoction, and were fed physiological saline, prednisone, or Sijunzi decoction, respectively. NMJ and muscle cell mitochondria ultrastructure were observed by transmission electron microscope. The amount of ATP was assessed by high performance liquid chromatography. The activities ofmitochondrial respiratory chain complexes I, Ⅱ, Ⅲ,and Ⅳ was determined using the Clark oxygen electrode method.RESULTS: In the model group, there were sparse muscle fibers, with decreased mitochondria, and sparse, diffluent, or absent NMJ folds. After intervention with Sijunzi decoction, the above pathology changes were improved: muscle fiber structure was clear and complete; the mitochondria count was higher; and the NMJ structure was close to normal. Gastrocnemius muscle mitochondria in the model group produced significantly less ATP than those in the prednisone group(P<0.01). Conversely, the ATP of Sijunzi decoction group was significantly higher than prednisone group(P<0.01). The activities of gastrocnemius muscle mitochondrial respiratory chain complexes I, Ⅱ, Ⅲ, and Ⅳ in both the prednisone and Sijunzi decoction groups was dramatically higher compared with the model group(P<0.05). The activities of complexes I and Ⅲ in the Sijunzi decoction group were significantly higher than those in the prednisone group(P<0.05), but there was no obvious difference in complex Ⅱ or Ⅳ activities between the two groups(P>0.05).CONCLUSION: Sijunzi decoction improved pathological changes in muscle mitochondria and NMJ,enhanced the amount of ATP in gastrocnemius muscle mitochondria, and improved the activities of respiratory chain complexes I, Ⅱ, Ⅲ, and Ⅳ(especially I and Ⅲ) of the EAMG rats.

Effects of herbal cake-partitioned moxibustion on the expression of thyroid autophagy-related factors LC3B and Beclin-1 in rats with autoimmune thyroiditis

Objective:To observe the anti-inflammatory effect,as well as the effect on the expression of microtubule-associated protein light chain 3B(LC3B)and Beclin-1 of herbal cake-partitioned moxibustion in rats with experimental autoimmune thyroiditis(EAT).Methods:Female Sprague-Dawley rats were randomly divided into a normal group and a modeling group.The EAT rat model was prepared by a combination of antigen immunization plus iodine agent induction.After the model was prepared,rats in the modeling group were randomly and equally divided into a model group and a herbal cake-partitioned moxibustion group.In the herbal cake-partitioned moxibustion group,moxibustion was alternately applied to two groups of points[Dazhui(GV14)-Mingmen(GV4)and Tiantu(CV22)-Guanyuan(CV4)],and the treatment continued for 30 d.Rats in the normal and model groups were only fixed identically without intervention.Histopathological manifestations of thyroid glands were observed by hematoxylin-eosin staining;the concentrations of thyroid peroxidase antibodies(TPOAb),thyroglobulin antibodies(TGAb),interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay;real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry were used to detect the mRNA and protein expression of autophagy-related factors LC3B and Beclin-1 in thyroid tissue.Results:There were massive follicular destruction,lymphocytic infiltration,and interstitial fibrous tissue hyperplasia of the thyroid glands in the model group.Some follicles of the thyroid glands were destroyed with few lymphocyte infiltrations and fibrous tissue hyperplasia in the moxibustion group.Compared with the normal group,the concentrations of serum TPOAb,TGAb,IL-1β,IL-6,and TNF-αwere increased in the model rats(P<0.05);the mRNA and protein expression levels of LC3B and Beclin-1 in thyroid tissue were reduced in the model group(P<0.05).Compared with the model group,the concentrations of serum TPOAb,TGAb,IL-1β,IL-6,and TNF-αwere reduced in the herbal cake-partitioned moxibustion group(P<0.05);the mRNA and protein expression levels of LC3B and Beclin-1 in thyroid tissue were increased in the herbal cake-partitioned moxibustion group(P<0.05).The mRNA and protein expression of LC3B and Beclin-1 in thyroid tissue was negatively correlated with the serum levels of TPOAb and TGAb.Conclusion:Herbal cake-partitioned moxibustion reduces the inflammatory response in the thyroid glands of EAT rats and lowers the levels of serum TPOAb and TGAb.This may be related to the regulation of mRNA and protein expression of the autophagy-associated factors LC3B and Beclin-1 in rat thyroid tissue.