Effect of cold stress on immunity in rats.

An increase in the morbidity of upper respiratory tract infections and the attack and exacerbation of autoimmune diseases has been observed to occur in the few days following sudden environmental temperature decreases, but the mechanisms for these phenomena are not well understood. To determine the effect of a sudden ambient temperature drop on the levels of stress hormones and T-lymphocyte cytokines in the plasma, the Toll-like receptor 4 (TLR4) expression of immunocompetent cells in rat spleens and the levels of regulatory T (Treg) cells in the peripheral blood, Sprague Dawley rats were divided into three groups of different ambient temperatures (20, 4 and -12°C). In each group, there were four observation time-points (1, 12, 24 and 48 h). Each ambient temperature group was subdivided into non-stimulation, lipopolysaccharide-stimulation and concanavalin A-stimulation groups. The levels of adrenocorticotropin (ACTH), epinephrine (EPI), angiotensin-II (ANG-II), interleukin-2 (IL-2), interferon-γ (IFN-γ), IL-4 and IL-10 in the plasma were determined using ELISA. The cellular expression levels of TLR4 and the presence of cluster of differentiation (CD)4+CD25+ and CD4+CD25+Forkhead box P3 (Foxp3)+ cells were determined using flow cytometry. The experiments demonstrated that the ACTH, EPI, ANG-II and IL-10 levels in the plasma were significantly increased at 4 and -12°C compared with those at 20°C, while the plasma levels of IFN-γ, IL-2 and IL-4, the TLR4 expression rates of immunocompetent cells in the rat spleen and the percentage of CD4+CD25+Foxp3+ Treg cells among the CD4+CD25+ Treg cells in the peripheral blood were decreased at 4 and -12°C compared with those at 20°C. These data indicate that cold stress affects the stress hormones and the innate and adaptive immunity functions in rats.

Effects of rhein on intestinal epithelial tight junction in IgA nephropathy.

AIM: To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). METHODS: Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7 per group): Control, IgAN, Rhein-treated, and Rhein-prevented. Bovine serum albumin, lipopolysaccharide and CCl4 were used to establish the rat model of IgA nephropathy. The Rhein-treated group was given rhein from week 7 until the rats were sacrificed. The Rhein-prevented group was given rhein from week 1. Animals were sacrificed at the end of week 10. We observed the changes in the intestinal epithelial tight junctions using transmission electron microscopy, and expression of intestinal epithelial tight junction proteins zona occludens protein (ZO)-1 and occludin by immunofluorescence using laser confocal microscopy. Changes in mRNA and protein expression of ZO-1 and occludin were measured by reverse transcriptase polymerase chain reaction and Western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography (HPLC) for assessing the intestinal permeability. RESULTS: In the control group, the tight junctions lied between epithelial cells on the top of the outer side of the cell membrane, and appeared in dense dotted crystal structures, the neighboring cells were binded tightly with no significant gap, and the tight junction protein ZO-1 and occludin were evenly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in the IgAN group, the structure of the tight junction became obscured and the dotted crystal structures had disappeared; the fluorescence of ZO-1 and occludin was uneven and weaker (5.37 ± 1.27 vs 10.03 ± 1.96, P < 0.01; 4.23 ± 0.85 vs 12.35 ± 4.17, P < 0.01); the mRNA expression of ZO-1 and occludin decreased (0.42 ± 0.19 vs 0.92 ± 0.24, P < 0.01; 0.40 ± 0.15 vs 0.97 ± 0.25, P < 0.01); protein expression of ZO-1 and occludin was decreased (0.85 ± 0.12 vs 1.98 ± 0.43, P < 0.01; 0.72 ± 0.15 vs 1.38 ± 0.31, P < 0.01); and the ratio of urinary lactulose/mannitol increased (3.55 ± 0.68 vs 2.72 ± 0.21, P < 0.01). In the Rhein-prevented and Rhein-treated groups, compared with the IgAN group, the intestinal epithelial tight junctions were repaired; fluorescence of ZO-1 and occludin was stronger (11.16 ± 3.52 and 8.81 ± 2.30 vs 5.37 ± 1.27, P < 0.01; 10.97 ± 3.40 and 9.46 ± 2.40 vs 4.23 ± 0.85, P < 0.01); mRNA of ZO-1 and occludin increased (0.81 ± 0.17 and 0.64 ± 0.16 vs 0.42 ± 0.19, P < 0.01; 0.82 ± 0.22 and 0.76 ± 0.31 vs 0.40 ± 0.15, P < 0.01); protein expression of ZO-1 and occludin was increased (2.07 ± 0.41 and 1.57 ± 0.23 vs 0.85 ± 0.12, P < 0.01; 1.34 ± 0.21 and 1.15 ± 0.17 vs 0.72 ± 0.15, P < 0.01); and the ratio of urinary lactulose/mannitol decreased (2.83 ± 0.43 and 2.87 ± 0.18 vs 3.55 ± 0.68, P < 0.01). CONCLUSION: Rhein can enhance the expression of ZO-1 and occludin, repair damaged tight junctions, and protect the intestinal barrier.

c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells.

AIM: To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells. METHODS: Human gastric cancer SGC-7901 cells were cultured in vitro. Following thermotherapy at 43°C for 0, 0.5, 1, 2 or 3 h, the cells were cultured for a further 24 h with or without the JNK specific inhibitor, SP600125 for 2 h. Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] and flow cytometry (Annexin vs propidium iodide). Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The production of p-JNK, Bcl-2, Bax and caspase-3 proteins was evaluated by Western blotting. The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction. RESULTS: The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy, and was 32.7%, 30.6%, 43.8% and 52.9% at 0.5, 1, 2 and 3 h post-thermotherapy, respectively. Flow cytometry analysis revealed an increased population of SGC-790l cells in G0/G1 phase, but a reduced population in S phase following thermotherapy for 1 or 2 h, compared to untreated cells (P < 0.05). The increased number of SGC-790l cells in G0/G1 phase was consistent with induced apoptosis (flow cytometry) following thermotherapy for 0.5, 1, 2 or 3 h, compared to the untreated group (46.5% ± 0.23%, 39.9% ± 0.53%, 56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%, P < 0.01), respectively. This was supported by the TUNEL assay (48.2% ± 0.4%, 40.1% ± 0.2%, 61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%, P < 0.01) respectively. More importantly, the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment (P < 0.01), and peaked at 2 h. A similar pattern was detected for Bax and caspase-3 proteins. Bcl-2 increased at 0.5 h, peaked at 1 h, and then decreased. Furthermore, the JNK specific inhibitor, SP600125, suppressed p-JNK, Bax and caspase-3 at the protein level in SGC790l cells following thermotherapy, compared to mock-inhibitor treatment, which was in line with the decreased rate of apoptosis. The expression of Bcl-2 was consistent with thermotherapy alone. CONCLUSION: Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels, and up-regulated the expression of Bax and caspase-3 proteins. Bcl-2 may play a protective role during thermotherapy. Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

[Experimental study on recombinant Bla g 2(rBla g 2) in the treatment of allergic asthma in mice].

OBJECTIVE: To study the therapeutic effect on murine allergic asthma with recombinant Bla g 2 (rBla g 2) allergen and its possible mechanism. METHODS: Eighteen BALB/C mice were randomly divided into three groups: normal control group (group A), asthma model group (group B), and recombinant protein rBlag2 treatment group (group C). Mice in groups B and C were subcutaneously immunized weekly with rBla g 2 (50 mg) formulated in Al (OH)3 adjuvant for three weeks. Group A received only adjuvant emulsified with normal saline. Two weeks after the last inoculation, mice in group C were administered each with rBla g 2 (100 mg) /dose, and groups A and B were given PBS. All the mice received eight doses at 2-day intervals. One week after the last immunotherapy, mice in groups B and C were intranasally challenged with 50 mg rBla g 2 daily for seven days, while mice in group A received PBS. Twenty-four hours after the challenge, the following items were examined: airway hyperresponsiveness of mice, total cellular score and cell classification in bronchoalveolar lavage fluid (BALF), level of rBla g 2-specific IgE and IgG2a in serum, lung inflammation by HE stain, and Bcl-2 expression of eosinophils of lung by immunohistochemistry. RESULTS: Compared with group B, group C showed a decreased Penh value of airway hyperresponsiveness (P < 0.05), reduced serum rBla g 2-specific IgE but increased IgG2a (P < 0.01), and reduced Bcl-2 expression of eosinophils. Total cells [(24.60 +/- 15.08) x 10(5)/ml] and eosinophils [(22.20 +/- 3.76) x 10(5)/ml] in BALF of group B significantly increased than those of group C [(14.30 +/- 4.95) x 10(5)/ml and (5.20 +/- 1.56) x 10(5)/ml, respectively] (P < 0.01). The interstitial space surrounding the airway lumen was characterized by a densely mixed cellular infiltrate, tissue edema and epithelium tissue damage in group B, while lung inflammation of group C reduced considerably. Each test value of group C was substantially similar to that of group A. CONCLUSION: The experiment shows proper immunotherapeutic efficacy of rBla g 2 in murine allergic asthma, which may possibly related to the apoptosis of eosinophils.

[Influence of induced nitric oxide synthase expression on apoptosis of thymocyte in burn rats].

OBJECTIVE: To investigate the influence of induced nitric oxide synthase (iNOS) expression on apoptosis of thymocyte in burn rats, and to explore the relationship between NO and pathological lesion of the thymus gland in burn rats. METHODS: Fifty-six male Wistar rats were enrolled in the study and randomized into control( C, n = 8,without treatment) , burn ( B, n = 24) , and S-methylisothiourea( SMT, n = 24) groups. Equal amount of isotonic saline solution and SMT(7. 5 mg/kg) were respectively intravenously infused into the rats in B and SMT groups after being inflicted with 30% TBSA full-thickness burns. The weight of thymus gland in each group were weighed, and thymocyte apoptosis and iNOS expression were determined with TUNEL method and immunohistochemistry, respectively at 6,24,72 postburn hours( PBH) , with 8 rats at each time-point. The number of apoptotic cells and the density of iNOS positive cells in thymus was measured by stereological method. RESULTS: The weight of thymus in B group at 24 and 72 PBH [ (153+/- 14) , (91+/-22) mg] were obviously heavier than those in C group, but much lighter than those in SMT group ( P < 0.01). A few apoptotic cells and iNOS positive cells were observed in cortex and medulla of thymus in C group, while they were observed in B group at 6 PBH, and the number of cells began to increase at 24 PBH, distributing in medulla,parenchyma, the boundary of cortex, and medulla under capsule. The iNOS positive cells in B group at 24 PBH were distributed around the interlobular septum. A large number of cortical cells with brown staining were observed in B group at 72 PBH, and the number of iNOS positive cells also increased, with scattered distribution and clear cell boundary. Fewer positive cells with uneven distribution, no iNOS positive cells, and few apoptotic foci were observed in SMT group after burns. The density of apoptotic cells in B group at 24 and 72 PBH was (2. 428 +/-0. 728) x 10(-5)/microm(3) and (5. 586 +/- 1.233) x 10(-5)/microm(3), respectively, which was obviously higher than that in C and SMT group. The density of iNOS positive cells in B group was increased in a time-dependent manner( P <0. 05). CONCLUSION: The apoptotic rate of thymocyte in severely burn rats increases early after burns. The up-regulation of iNOS expression in thymus can promote apoptosis of thymocytes, while SMT can partially ameliorate this phenomenon.

[Ultrastructural changes in the midgut epithelium of Ixodes sinensis after infesting the rabbits immunized by purified ixodic protein].

OBJECTIVE: To observe the ultrastructural changes in the midgut epithelium of Ixodes sinensis after infesting rabbits immunized with Mr 105000 purified tick antigen. METHODS: New Zealand rabbits were inoculated with Mr 105000 purified antigen by means of mutiple intradermal injection in foot pad, groin and back. Each immunized rabbit was infested by 30 female Ixodes sinensis. At 24 hours, 48 hours, 72 hours, 5 days and 8 days after infestation, three Ixodes sinensis in each group were observed for ultrastructural changes in the epithelium of their midgut. RESULTS: Histological examinations showed that with the time going, digestive cells of the ticks after infesting hosts became more and larger with dense and regularly arranged microvilli, enriched organella, distinct unit-membrane structure, and the appearance of tubli, small vacuole, numerous lipid droplets and hematin granules. These cells also developed a highly infolded basal lamina, forming a labyrinth system. The digestive cells of immunized group were however greatly damaged, whose number and volume were significantly different from control groups. From 24 to 48 hours after infestation, the midgut epithelium of Ixodes snenss showed pathological changes with the basal lamina becoming thinner, looser and broken; digestive cells damaged and vacuolated; microvilli decreased, shortened and irregularly arranged; the mitochondria swollen and its crests reduced, shortened and even with myeloid changes; the rough endoplasmic reticulum dilated; lipid droplets and hematin granules decreased; phagocytic and pinocytic activity weakened; and basal labyrinth system vacuolated. From 72 hours to 8 days after infestation, cells were severely damaged, organella were denatured and necrotic, nuclei showed pyknosis and cells lysed. CONCLUSION: The rabbits immunized with Mr 105000 purified ixodic protein have acquired the adoptive immunity against Ixodes sinensis; in the anti-tick immunity described above, the midgut of Ixodes sinensis is the major affected site.