Louki Zupa decoction attenuates the airway inflammation in acute asthma mice induced by ovalbumin through IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway.

CONTEXT: Asthma is a common respiratory system disease. Louki Zupa decoction (LKZP), a traditional Chinese medicine, presents a promising efficacy against lung diseases. OBJECTIVE: To investigate the pathogenic mechanism of asthma and reveal the intervention mechanism of LKZP. MATERIALS AND METHODS: Forty-eight female Balb/c mice were randomly divided into 6 groups: normal control group (NC), ovalbumin (OVA)/saline asthma model group, OVA/LL group, OVA/LM group, OVA/LH group and OVA/DEX group (n = 8 per group). The asthmatic mice were modelled through intraperitoneal injecting and neutralizing OVA. LKZP decoction was administrated by gavage at the challenge stage for seven consecutive days (2.1, 4.2 and 8.4 g/kg/day). We investigated the change in lung function, airway inflammation, mucus secretion and TH-1/TH-2-related cytokines. We further verify the activated status of the IL-33/ST2/NF-κB/GSK3ß/mTOR signalling pathway. RESULTS: LKZP was proved to improve asthmatic symptoms, as evidenced by the down-regulated airway resistance by 36%, 58% and 53% (p < 0.01, p < 0.001 vs. OVA/saline group), up-regulated lung compliance by 102%, 114% and 111%, decreased airway inflammation and mucus secretion by 33%, 40% and 33% (p < 0.001 vs. OVA/saline group). Moreover, the content of cytokines in BALF related to airway allergy (such as IgE) and T helper 1/T helper 2 cells (like IL-2, IL-4, IL-5, IL-13, TNF-α and IFN-γ), were also markedly reduced by 13-65% on LKZP intervention groups compared with model group. Mechanistic research revealed that the IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway was activated in the OVA/saline group and LKZP significantly down-regulated this pathway. DISCUSSION AND CONCLUSION: LKZP improves lung function, airway inflammation, mucus secretion and correct immune imbalance by intervening with the IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway, presenting a promising therapeutic choice for asthma.

[The Study of Lipofibroblasts Differentiation of CD90⁺ and CD90⁻ Orbital Fibroblasts Subsets in Patients with TAO].

OBJECTIVE: Divided orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) into CD90⁺ and CD90⁻ subsets respect to surface CD90 expression, then determined whether CD90⁺ and/or CD90⁻ fibroblasts were capable of differentiating into lipofibroblasts. METHODS: Fibroblasts subset separation into CD90⁺ and CD90⁻ subsets was accomplished by three to four rounds of magnetic bead selection, then treated with 3-isobutyl-1-lmethylxanthine (IBMX), insulin and dexamethasone which was an known inducer of the lipofibroblastic phenotype, then the cells were observed every day to find Lipid droplets. Oil red O staining were conducted at 5 d, 10 d, 15 d, 20 d and 25 d after inducing. The percent of lipofibroblasts were calculated. RESULTS: The ratio in fibroblast derived from extraocular muscles of differentiating into lipofibroblast is less than from connective/adipose tissue (P < 0.05). The ratio of CD90⁺ fibroblast is less than CD90⁻ fibroblast (P < 0.05). CD90⁺ cells derived from extraocular miscles could not be induced to differentiate into lipofibroblast. The ratio in CD90⁻ fibroblast from connective/adipose tissue is highest (P < 0.05). CONCLUSION: Orbital fibroblast which has the function of differentiating into lipofibroblast is mainly CD90⁻ connective/adipose tissue.

Preventive effect of pidotimod on reactivated toxoplasmosis in mice.

As one of food-borne parasitic diseases, toxoplasmosis entails the risk of developing reactivation in immunocompromised patients. The synthetic dipeptide pidotimod is a potent immunostimulating agent that improves the immunodefenses in immunodepression. To investigate the efficacy of pidotimod as a preventive treatment, we used a murine model of reactivated toxoplasmosis with cyclophosphamide (CY)-induced immunosuppression. Pidotimod administration significantly restored the body weight and spleen organ index, increased survival time (from 70 to 90%), and decreased the parasitemia (from 80 to 35%) of CY-induced mice with reactivated toxoplasmosis. Cytokine profiles and CD4(+) T cells subpopulation analyses by Cytometric Bead Array and flow cytometry demonstrated that pidotimod treatment resulted in a significant upregulation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th1 cells (from 3.73 ± 0.39 to 5.88 ± 0.46%) after CY induction in infected mice. Additionally, histological findings and parasite DNA quantification revealed that mice administered with pidotimod had a remarkable reduction of parasite burden (two-log) and amelioration of histopathology in the brains. The in vitro studies showed that pidotimod significantly restored concanavalin A-induced splenocyte proliferation and pro-inflammatory cytokines in the supernatants of splenocyte culture. It could be concluded that the administration of pidotimod in immunocompromised mice significantly increases the Th1-biased immune response, prolongs survival time, and ameliorates the load of parasites in the blood. This is the first report of the preventive effect of pidotimod on reactivated toxoplasmosis.

[Study on the characteristics of optic nerve meningiomas in medical imageology,histopathology and immunohistochemistry].

OBJECTIVE: To determine the characteristics of optic nerve meningiomas in medical imageology (including CT and MRI), histopathology and immunohistochemical expression of Vimentin, CK, S-100, EMA in tumor cells. METHODS: This was a retrospective study on a serial of clinical cases. Forty-eight cases were collected from the past 21 years at the Department of Ophthalmology, West China Hospital, Sichuan University. All the cases underwent surgery and were confirmed as optic nerve meningiomas by histopathological test, including paraffin imbedded sectioning and HE staining. In addition, all the cases had medical records on CT and 17 cases had MIR scan. Immunohistochemical staining for VIMENTIN, CK, S-100, EMA was performed in the 21 cases. RESULTS: Characteristics of the CT scan include that, in 39 cases, among them 26 cases the tumors filled in the orbit especially at the orbital apex, which led to the disappearance of the black triangle. Three cases showed enlargement and calcification of optic nerve and therefore train track sign was seen. Three cases showed that the masses of optic nerve were close to the globe and another three cases showed that the masses were located in the peripheral orbit. Thirteen of 17 cases MRI disclosed the big orbital tumors and 5 cases could better show tumor extension to optic cross or intracranium. Histopathological tests demonstrated meningothelial or syncytial type in 39 cases, transitional or mixing type in 8 cases, fiber or fibroblast type in 3 cases and vascular type in 2 cases. Immunohistochemistry study verified the positive staining rate of VIMENTIN as 90.5%(21 cases), EMA 66.7%(15 cases), CK 42.8%(9 cases) and S-100 23.8%(5 cases), respectively. CONCLUSIONS: Characteristics of CT and MRI include enlargement and tumor-like expansion of optic nerve. Most of the tumors reach to orbital apex and show uneven density. Calcification may occur in the tumor. The main pathological type of the tumors is meningothelial. Positive immunohistochemiscal staining for VIMENTIN and EMA may be helpful in diagnosis.

Role of cellular senescence in inflammatory lung diseases.

Cellular senescence, a characteristic sign of aging, classically refers to permanent cell proliferation arrest and is a vital contributor to the pathogenesis of cancer and age-related illnesses. A lot of imperative scientific research has shown that senescent cell aggregation and the release of senescence-associated secretory phenotype (SASP) components can cause lung inflammatory diseases as well. In this study, the most recent scientific progress on cellular senescence and phenotypes was reviewed, including their impact on lung inflammation and the contributions of these findings to understanding the underlying mechanisms and clinical relevance of cell and developmental biology. Within a dozen pro-senescent stimuli, the irreparable DNA damage, oxidative stress, and telomere erosion are all crucial in the long-term accumulation of senescent cells, resulting in sustained inflammatory stress activation in the respiratory system. An emerging role for cellular senescence in inflammatory lung diseases was proposed in this review, followed by the identification of the main ambiguities, thus further understanding this event and the potential to control cellular senescence and pro-inflammatory response activation. In addition, novel therapeutic strategies for the modulation of cellular senescence that might help to attenuate inflammatory lung conditions and improve disease outcomes were also presented in this research.

Apigenin ameliorates non-eosinophilic inflammation, dysregulated immune homeostasis and mitochondria-mediated airway epithelial cell apoptosis in chronic obese asthma via the ROS-ASK1-MAPK pathway.

BACKGROUND: Obese asthma is one of the important asthma phenotypes that have received wide attention in recent years. Excessive oxidative stress and different inflammatory endotypes may be important reasons for the complex symptoms, frequent aggravation, and resistance to traditional treatments of obese asthma. Apigenin (API), is a flavonoid natural small molecule compound with good anti-inflammatory and antioxidant activity in various diseases and proved to have the potential efficacy to combat obese asthma. METHODS: In vivo, this study fed C57BL/6 J mice with high-fat diets(HFD)for 12 weeks and then stimulated them with OVA for 6 weeks to establish a model of chronic obese asthma, while different doses of oral API or dexamethasone were used for therapeutic interventions. In vitro, this study used HDM to stimulate human bronchial cells (HBEs) to establish the model and intervened with API or Selonsertib (SEL). RESULTS: This study clarified that OVAinduced a type of mixed granulocytic asthma with elevated neutrophils and eosinophils in obese male mice fed with long-term HFD, which also exhibited mixed TH17/TH1/TH2 inflammation. Apigenin effectively suppressed this complex inflammation and acted as a regulator of immune homeostasis. Meanwhile, apigenin reduced AHR, inflammatory cell infiltration, airway epithelial cell apoptosis, airway collagen deposition, and lung oxidative stress via the ROS-ASK1-MAPK pathway in an obese asthma mouse model. In vitro, this study found that apigenin altered the binding status of TRAF6 to ASK1, inhibited ASK1 phosphorylation, and protected against ubiquitin-dependent degradation of ASK1, suggesting that ROS-activated ASK1 may be an important target for apigenin to exert anti-inflammatory and anti-apoptotic effects. To further verify the intervention mechanism, this study clarified that apigenin improved cell viability and mitochondrial function and inhibited apoptosis by interfering with the ROS-ASK1-MAPK pathway. CONCLUSIONS: This study demonstrates for the first time the therapeutic effect of apigenin in chronic obese asthma and further clarifies its potential therapeutic targets. In addition, this study clarifies the specificity of chronic obese asthma and provides new options for its treatment.

[CCR1 expression on immune cells in orbital tissue of patients with thyroid associated ophthalmopathy].

OBJECTIVE: To investigate the expression of CC chemokine receptor 1(CCR1) on the immune cells in orbital tissue of patients with thyroid associated ophthalmopathy(TAO). METHODS: The tissue samples of orbital tissue were collected from 25 cases of TAO patient and 10 normal control subjects, and the expression of CCR1 in the immune cells was studied by SP immunohistochemistry. The correlation between CCR1 and clinical activity scores (GAS) was analyzed. RESULTS: The expression of CCR1 on immune cells was positive, in orbital tissue of patients with TAO, and the positive rate was 84%, while it was negative or low positive in normal control subjects. The difference between them was significant (P < 0.05). In addition, the expression of CCR1 on the immune cells of orbital tissue with TAO had significant correlation with CAS (P < 0.05). CONCLUSION: The expression of chemokine receptor CCR1 on the immune cells in orbital tissue of TAO patients is higher than that of normal subjects. CCR1 may play a role in the pathogenesis of TAO.

[Kinetic distribution of cyst-forming Chinese isolate of Toxoplasma gondii in mice].

OBJECTIVE: To determine the kinetics of infection and cyst formation in CD1 mice following oral infection with cyst-forming Chinese isolate of Toxoplasma gondii TgCtwh1(genotype China 1, ToxoDB#9). METHODS: 50 CD1 female mice were obtained from specific pathogen-free (SPF) mouse colony in the Vital River Laboratories (VRL), Beijing. Mice were randomly divided into 10 groups each with 5 mice. All mice but control were peroral gavage infected with 50 cysts (1x10(4) bradyzoites) of TgCtwh1 isolate of T. gondii isolated from Wuhan, China. Cysts were isolated from the entire brain of mice infected with TgCtwh1 by density gradient centrifugation over Fycoll-paque plus. Animals were orally inoculated with cysts on day zero, and peripheral blood, lymph nodes, heart, liver, and brain of infected mice were collected on days 2, 4, 7, 10, 14, 21, 35, 50, and 72 post infection. Five mice were sacrificed by cervical dislocation under anesthesia at each time of collection, and the kinetic distribution was detected by fluorescence quantitative PCR and tissue inoculation into fresh mice. The cyst formation at various intervals after infection was also observed, as was the number of the cysts in brains and the cyst-forming rate. RESULTS: The body weight of the mice lessened (3.650 +/- 0.252)g post oral infection on day 7, and the weight was progressively decreased between day 10 [(1.730 +/- 0.017)g] and day 14 [(-0.390 +/- 0.554) g] after infection (P<0.05). In the brain tissue, cysts were first observed on day 21 post oral infection and the cyst-forming rate was 80%, and the average diameter of cysts was 20-40 microm. While on day 35 after infection, the cysts were formed in all infected mice(cyst-forming rate was 100%) and the average diameter was 50-60 microm. In chronic infection, DNA copies of parasites were first detected in blood, heart, liver and lymph node at 3.51 +/- 0.152, 4.100 +/- 0.198, 4.220 +/- 0.209 and 4.960 +/- 0.052 respectively on day 2, then in the brain on day 4 (3.800 +/- 0.154). During the early days of infection, the parasite burden in blood was progressively increased until days 7 (5.240 +/- 0.115) then gradually decreased and become undetectable on day 35. The burden of T. gondii in the heart and brain tissues increased significantly and reached their maximum on day 14 (5.640 +/- 0.214) and day 10 (5.790 +/- 0.060), respectively, and remained a stable level thereafter. Liver and lymph tissues reached their maximum on day 7 (5.310 +/- 0.038) and day 10 (6.200 +/- 0.152), then gradually decreased and become undetectable on day 50. CONCLUSION: The parasitemia in mice infected with T. gondii cyst-forming isolate lasts for 21 d at least, and cysts are detected in brain on day 21.

[Expression of CCR1 in the orbital fibroblasts with thyroid associated ophthalmopathy].

OBJECTIVE: To investigate the expression of chemokine receptor (CCR1) in the orbital fibroblasts (OFs) with thyroid associated ophthalmopathy (TAO) and to explore the pathogenesis of TAO. METHODS: By immunohistochemistry, the expression of CCR1 was studied in 25 cases of TAO patients and 10 normal control subjects and the correlation between CCR1 and GAS was analysed. RESULTS: The expression of CCR1 in the OFs with TAO was positive, but it was negative or weak positive in normal control subjects. The difference between them was significant (P < 0.05). And the expression of CCR1 in the OFs with TAO had no significant correlation with GAS (r = 0. 048, P > 0.05). CONCLUSION: The expression of chemokine receptor CCR1 is higher on OFs of TAO patients than that of normal subjects. CCR1 maybe play a role in pathogenesis of TAO.

Evaluation of recombinant SjLAP and SjFBPA in detecting antibodies to Schistosoma japonicum.

OBJECTIVE: To investigate the early response of immunoglobulin G (IgG) antibody responses to Schistosoma japonicum infection in mice by using the recombinant proteins, S. japonicum leucine aminopeptidase (rSjLAP) and S. japonicum fructose-1, 6-bisphosphate aldolase (rSjFBPA), and evaluate the potential of rSjLAP and rSjFBPA in diagnosis as well as in assessment of therapeutic efficacy in human schistosomiasis. METHODS: rSjLAP or rSjFBPA was induced from Escherichia coli BL21 strain transfected with the expression vectors, pET-28a-rSjFBPA/BL21 or pET-28a-rSjLAP/BL21 using isopropyl-beta-D-thiogalactoside (IPTG), and purified by Ni-NTA His Bind resin. 88 BALB/c female mice, inbred and 6 to 8 weeks old, were randomly divided into 4 groups. Groups A, B and C each made up of 21 mice and group D comprised 25 mice. Groups A, B and C were infected with 5, 15 and 25 S. japonicum cercariae respectively. As control, mice in group D were left uninfected. 3 mice from each of groups A, B and C were sacrificed and sera collected on days 3, 7, 10, 14, 20, 30, and 60 post infection. All the 25 mice in group D were sacrificed on the first day of the experiment for serum collection. rSjLAP and rSjFBPA were screened and used in ELISA to test the antibody response of the serum samples. Also, sera of 38 acute patients, 96 chronic patients with schistosomiasis japonica, 90 healthy donors and patients with other parasite infections including Clonorchis sinensis (33 cases), Paragonimus westermani (40) and hookworms (37) were tested using the recombinant protein-based ELISA. In addition, 36 sera each from the acute and chronic patients 12 months after treatment with praziquantel and 64 of the chronic patients in more than 2 years post-treatment of praziquantel were tested. The dosage of praziquantel for both acute and chronic patients was 60 mg/kg, 2 times/dx2 d. RESULTS: IgG antibody response was first detected at day 10 post infection by rSjLAP, rSjFBPA or the combined antigen assay. The mean absorbance (A450) on this day were 0.535 +/- 0.053, 0.595 +/- 0.033, 0.696 +/- 0.104 for group B; 0.548 +/- 0.060, 0.608 +/- 0.063, 0.621 +/- 0.090 for group C; and 0.415 +/- 0.038, 0.455 +/- 0.056, 0.498 +/- 0.077 for group A for rSjLAP, rSjFBPA and the combined assay respectively (P < 0.05). Early antibody level to both antigens was significantly higher in mice infected with 15 or 25 cercariae than those with 5 cercariae (P < 0.05). However, ELISA results in patients with confirmed schistosomiasis revealed positive rates of 97.4% (37/38) and 87.5% (84/96) for acute and chronic schistosomiasis with rSjLAP , 94.7% (36/38) and 88.5% (85/96) for acute and chronic schistosomiasis with rSjFBPA and 94.7% (36/38)and 85.4%(82/96) with both rSjLAP and rSjFBPA respectively. Statistical analysis showed no significant difference in the positive rate (P > 0.05). Also, rSjLAP and combined antigens showed a specificity of 96.7% (87/90) while that of rSjFBPA was 97.8% (88/90). There was a general decrease in the antibody titer of the patients after treatment. In 12 months after treatment it was 0.236 +/- 0.212 with rSjLAP, 0.287 +/- 0.191 with rSjFBPA, and 0.235 +/- 0.120 with both antigens respectively for acute cases; For chronic patients, it was 0.266 +/- 0.124, 0.261 +/- 0.143 and 0.265 +/- 0.140 in 12 months post-treatment, and 0.204 +/- 0.074, 0.176 +/- 0.074, and 0.176 +/- 0.073 in 2 years, respectively. For healthy control, it was 0.188 +/- 0.056, 0.173 +/- 0.45, and 0.184 +/- 0.051, respectively. No significant difference on antibody titer was found between treated patients and control (P > 0.05). The cross reaction with C. sinensis was 15.2% (5/33) for rSjLAP, 12.1% (4/33) for rSjFBPA and 9.2% (3/33) for combined antigens. With P. westermani, it was 15.0% (6/40), 12.5% (5/40) and 15.0% (6/40), respectively, and 8.1% (3/37) with hookworm infection. CONCLUSION: The study showed a satisfactory sensitivity and specificity of rSjLAP and rSjFBPA by ELISA which is promising for the immunological diagnosis of schistosomiasis.

Role of src-suppressed C kinase substrate in rat pulmonary microvascular endothelial hyperpermeability stimulated by inflammatory cytokines.

OBJECTIVE: The aim of the study was to investigate the role of src-suppressed C kinase substrate (SSeCKS) in the modulation of rat pulmonary microvascular endothelial cells (RPMVEC) permeability elicited by interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. METHODS: The gene expression of SSeCKS was analyzed by reverse transcription-polymerase chain reaction. Immunoblotting was used to determine the SSeCKS protein expression and the activation of the protein kinase C (PKC) signaling pathway. A RPMVEC monolayer was constructed to determine changes of transendothelial electrical resistance (TER) and FITC-dextran flux (P (d)) across the monolayer. SSeCKS-specific small interfering RNA was transfected into RPMVEC. RESULTS: IL-1ß and TNF-α activated the PKC signaling pathway in RPMVEC, and up-regulated the gene and protein expression of SSeCKS. Depletion of endogenous SSeCKS in RPMVEC significantly attenuated cytokine-induced decrease in TER and increase in P (d), but not to the basal levels. PKC inhibitors also significantly decreased cytokine-induced hyperpermeability and SSeCKS expression. CONCLUSIONS: SSeCKS is involved in the endothelial hyperpermeability induced by IL-1ß and TNF-α in inflammatory process.

[Cloning and expression of rotavirus SA11 VP7 and preparation of IgY antibodies against recombinant VP7].

OBJECTIVE: To prepare eukaryotic expression of rotavirus (RV) SA11 capsid protein VP7, and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens. METHODS: MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomic RNA of RV SA11. The PCR products were ligated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZalphaB. The recombinant pPICZalphaB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and transformed into Pichia pastoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7. RESULTS: The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZalphaB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western blotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10.2 mg per egg. CONCLUSION: The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.

[Amiota magna as an intermediate host of Thelazia callipaeda under laboratory conditions].

A number of flies around the eyes of a person or around a fruit bait were collected from Huangshan Mountain, and experimentally infected by newborn larvae of Thelazia callipaeda. After 20 days, the flies were examined for T. callipaeda. Following dissection, 3 (30%, 3/10) of Amiota magna, and 55 (21.6%, 55/255) of A. okada were found infected by T. callipaeda. The susceptibility of T. callipaeda is similar in the two species fruit flies (chi2=0.0584, P> 0.05). The rabbits were infected by infective larvae of T. callipaeda from A. magna. At the 35th day after infection, the newborn larvae and worms of T. callipaeda were found in the conjunctival sac of rabbits. This study suggested that A. magna acts as intermediate host of T. callipaeda under laboratory conditions.

Toxoplasma ROP16I/III ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease. AIM: To explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells. METHODS: RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) (M1 cells) were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro. The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16 I/III. The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, transforming growth factor (TGF)-ß1, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) was detected. The expression of iNOS, Arg-1, signal transducer and activator of transcription 3 (Stat3), p-Stat3, Stat6, p-Stat6, programmed death ligand-2 (PD-L2), caspase-3, -8, and -9 was analyzed by Western blotting, and Griess assays were performed to detect nitric oxide (NO). TNF-α, IL-1ß, IL-6, TGF-ß1, and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay, and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system. RESULTS: M1 cells exhibited significantly increased production of iNOS, NO, TNF-α, IL-1ß, and IL-6, while ToxoROP16I/III induced macrophage bias to M2 cells in vitro, showing increased expression of Arg-1, IL-10 and TGF-ß1 and elevated production of p-Stat3 and p-Stat6. The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2. Accordingly, Caco-2 cells became apoptotic, and apoptosis-associated proteins such as caspase-3, -8 and -9 were dampened during co-culture of M1 and M2 cells. Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells, but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis. CONCLUSION: ToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages. This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA.

AIM: To investigate the inhibitory effects of hepatitis B virus (HBV) replication and expression by transfecting artificial microRNA (amiRNA) into HepG2.2.15 cells. METHODS: Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by fluorescence quantitative PCR, and the level of HBV S mRNA was measured by semi-quantitative RT-PCR. RESULTS: The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection (P < 0.01 for all). The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% +/- 4.7% and 39.9% +/- 6.7%, respectively, at 72 h in amiRNA-HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection (P < 0.01 for all). In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups (P < 0.01 for all, vs negative control). The copies of HBV DNA were inhibited by 33.4% +/- 3.0%, 60.8% +/- 2.3% and 70.1% +/- 3.3%, respectively. CONCLUSION: In HepG2.2.15 cells, HBV replication and expression could be inhibited by artificial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.

[Effects of rh-leptin on the proliferation and migration of orbital preadipocytes of thyroid associated ophthalmopathy].

OBJECTIVE: To investigate the effects of rh-leptin on the proliferation, migration of orbital preadipocytes of thyroid associated ophthalmopathy (TAO). METHODS: Preadipocytes were cultured from orbital tissues of TAO patients after orbital decompression. The subcultured preadipocytes were incubated with different concentrations of rh-leptin. The proliferation of preadipocytes was tested by methyl thiazlyl tetrazolium (MTT) assay, and the migration was evaluated by crutch method. RESULTS: With the treatment of 500 mg/L rh-leptin, the preadipocytes proliferated more than control (P<0.05), while 10-100 mg/L rh-leptin has no such significant effects. The migration of the preadipocytes was enhanced by rh-leptin, which showed statistic difference in 50-500 mg/L rh-leptin groups when compared with control group (P<0.05). CONCLUSION: Rh-leptin could improve the proliferation and migration of TAO orbital preadipocytes in vitro, which suggests that leptin may play an important role in the pathogenesis of TAO.

[Clinicopathologic observation of orbital natural killer-T cell lymphoma].

OBJECTIVE: To investigate clinical manifestation, diagnosis and treatment of orbital natural killer (NK)-T cell lymphoma. METHODS: It was a retrospective case series. Seven orbital NK-T cell lymphoma patients confirmed by surgical biopsies were collected during the past 22 years. We reviewed the records, surgical and treatment procedures. Surgical specimens were studied with HE staining, immunohistochemical staining and molecular biological analysis. RESULTS: These patients had proptosis, eye motive inhibition or fixation and visual acuity was decreased or even without light perception. Skin of inner canthus and eyelids appeared red and swollen, with ulceration and cavity formation. CT scan revealed that the tumor showed uneven density and an unclear border. Tremendous lymphocyte infiltration and tissue necrosis in the tumor were observed in the biopsy tissue. LCA, CD45RO and CD57 immunohistochemical staining revealed positive results. Clonal T-cell-receptor gene rearrangements of two patients showed negative results and the Epstein-Barr virus was detected. CONCLUSIONS: Orbital NK-T cell lymphoma is a rare disease. The characteristics of this disease include a highly aggressive clinical course, severe destruction and a poor prognosis. The final diagnosis depends on HE staining, immunohistochemical staining and molecular biological examination.

[Effect of paeoniflorin on secretion of TGF-beta1 from macrophages in mice].

OBJECTIVE: To explore the effect of paeoniflorin (PAE) on the production of transforming growth factor beta1 (TGF-beta1) from peritoneal macrophages(PMs) stimulated by soluble egg antigen (SEA) of Schistosoma japonicum. METHODS: SEA was prepared by trituration and added into culture plank, flask and dish containing PMs which were cultured for 24 h. TGF-beta1 secreted from PMs was measured by ELISA. TGF-beta1 mRNA and protein produced from PMs were evaluated by RT-PCR and Western blotting, respectively. SEA (10 mg/L) 5 ml was added into culture flask and dish containing PMs. PMs were cultured for 12 h, and PAE at different concentrations (0, 7.5, 15, 30, 60, 120 mg/L) was added into the culture flask and dish, and PMs were cultured consecutively for another 12 h and 24 h, respectively. TGF-beta1 mRNA and protein from PMs stimulated by SEA were evaluated by RT-PCR and Western blotting, respectively. RESULTS: TGF-beta1 (235.86 +/- 3.43 ng/L) was produced from PMs under stimulation of SEA at 10 mg/L, and the expression of TGF-beta1 mRNA and protein in PMs were depressed significantly by PAE in a concentration-dependent manner (r = -0.827, P < 0.01; r = -0.952, P < 0.01, respectively). CONCLUSION: PAE inhibits the production of TGF-beta1 from PMs stimulated by SEA.

[Effect of paeoniflorin on hepatic immunopathogenesis in mice with Schistosoma japonicum infection].

OBJECTIVE: To investigate the mechanism of paeoniflorin in preventing hepatic granuloma formation and fibrosis in mice infected with Schistosoma japonicum. METHODS: Model of hepatic granuloma and fibrosis was established by infecting mice with S. japonicum cercariae. The infected mice were randomly divided into 4 groups: group A as model (infected control) group (15 mice), and paeoniflorin being given before, simultaneously and after praziquantel treatment as groups B, C and D. Each of the groups B, C and D was subdivided into 3 subgroups (15 mice each): low dose (paeoniflorin 2 ml, 30 mg/(kg x d) x 30 d), high dose(paeoniflorin 2 ml, 120 mg/(kg x d) x 30 d) and control (2 ml, 0.5% sodium carboxymethylcellulose x 30 d). In group B, paeoniflorin or sodium carboxymethylcellulose was orally administrated on 12 d after infection. In groups C and D, paeoniflorin or sodium carboxymethylcellulose was administrated on 42 d or 72 d after infection. Each of group B, C and D was orally given praziquantel 2 ml (500 mg/(kg x d) x 2 d) on 42 d after infection. On the 102nd day after infection, all animals were sacrificed by cervical dislocation. Serum hyaluronic acid (HA) was detected by radioimmunoassay; area of egg granuloma and degree of hepatic fibrosis were observed via HE and Masson stainings; the expression of transforming growth factor beta1 (TGF-beta1), alpha smooth muscle actin (alpha-SMA and collagen I (Col I) protein were measured by immunohistochemical method. RESULTS: In group B, the level of HA (0.719 +/- 0.239 microg/ml, 0.721 +/- 0.182 microg/ml) in low or high dose subgroups was significantly lower (F = 9.429, P < 0.01) than the control subgroup (1.049 +/- 0.286 microg/ml); the area of granuloma (0.066 +/- 0.005 mm2, 0.064 +/- 0.004 mm2) or the degree of hepatic fibrosis (2.067 +/- 0.458, 1.967 +/- 0.399) in low or high dose subgroups was significantly greater (F = 862.540, F = 29.738, P < 0.01) than the control (0.141 +/- 0.008 mm2, 3.467 +/- 0.834); the expression of alpha-SMA positive cells (2.933 +/- 0.594, 3.000 +/- 0.535) in low or high dose subgroups was significantly lower (F = 12.323, P < 0.01, P < 0.01) than its control (4.800 +/- 1.859); the expression of TGF-beta1 (0.256 +/- 0.057, 0.274 +/- 0.054) in low or high dose subgroups was significantly lower (F = 148.990, P < 0.01) than its control (0.552 +/- 0.047); the content of Col I (0.334 +/- 0.041, 0.339 +/- 0.042) in low or high dose subgroups was significantly lower (F = 180.881, P < 0.01) than its control (0.601 +/- 0.049). In groups C & D, no significant difference was found between the low or high dose subgroups or between the subgroups and their corresponding controls. CONCLUSION: Paeoniflorin can significantly reduce hepatic granuloma formation and fibrosis due to schistosome eggs, and decrease the expression of TGF-beta1, alpha-SMA in mice when it is given before praziquantel administration, which may associate with the activation of hepatic stellate cells and the expression of TGF-beta1 in liver tissue.

[Effect of dexamethasone on expression of intercellular adhesion molecule-1 in orbital fibroblasts from thyroid associated ophthalmopathy].

OBJECTIVE: To investigate the effects of dexamethasone (DEX) on the expression of intercellular adhesion molecule-1 (ICAM-1) in orbital fibroblasts (OF) from patients with thyroid associated ophthalmopathy (TAO). METHODS: OFs from 10 patients with TAO and from 5 normal control subjects were cultured in vitro. OFs were incubated with DEX, IFN-gamma or DEX and IFN-gamma compound for 72 hours. Flow cytometry analyses were performed to determine ICAM-1 expression. RESULTS: Baral ICAM-1 expression in OFs from TAO was significantly higher than that in the controls. DEX (10(-6) mol/L) inhibited expression of ICAM-1. IFN-gamma (300 U/ mL) stimulated ICAM-1 expression significantly, which was inhibited by DEX (10(-6) mol/L). CONCLUSION: ICAM-1 plays an important role in the pathogenesis of TAO. DEX not only inhibits ICAM-1 expression, but also inhibits IFN-gamma-induced ICAM-1 expression on cultured OF in vitro. This may be part of the mechanism by which glucocorticoid has efficacy in the treatment of TAO.