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Objective:To investigate the effect of Danggui Yinzi on allergic reaction in chronic urticaria (CU) mice model and the mechanism of autophagy intervention. Method:The SPF BALB/c mice were used to replicate the CU mice model by intraperitoneal injection of ovalbumin and aluminum hydroxide suspension. The animals were randomly allocated into six groups: a normal group (normal saline 20 mL·kg-1·d-1), a model group (normal saline 20 mL·kg-1·d-1), a loratadine group(0.001 3 g·kg-1·d-1), a Danggui Yinzi high，medium and low-dose group（39.3，19.6，9.8 g·kg-1·d-1）. The pathological changes of skin tissues were observed by hematoxylin-eosin (HE) staining. Morphological changes of autophagy in skin tissues epithelial cells were observed by transmission electron microscope. The mRNA levels of microtubule-associated protein 1 light chain 3B(LC3B) and ubiquitin-binding protein p62 mRNA in skin tissues were detected by real-time quantitative polymerase chain reaction (Real-time PCR). The expressions of LC3B and p62 in skin tissues were detected by immunohistochemistry (IHC). Result:Danggui Yinzi can significantly improve the pathological manifestations of dermal edema, collagen bundles separation, telangiectasia in CU mice， it can also improve autophagosomes formation and abnormal cell ultrastructure such as nuclear chromatin condensation, mitochondrial swelling, endoplasmic reticulum expansion, etc. Compared with the normal group, the protein expressions of LC3B in skin tissues of the model group was significantly increased (P<0.01), LC3B mRNA level was increased too, while p62 mRNA levels and its protein expressions were decreased-regulated (P<0.01). Compared with the model group, levels of LC3B mRNA and protein expressions of the Danggui Yinzi groups were significantly increased (P<0.05,P<0.01), while p62 mRNA levels and its protein expressions were significantly decreased-regulated (P<0.05,P<0.01). Conclusion:Danggui Yinzi can regulate the expression of LC3B, p62 mRNA and protein expressions, enhance the level of autophagy, and improve the pathological state of CU mice.
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Objective To investigate the relationship between different tidal volume (VT) mechanical ventilation (MV) and autophagy and mitochondrial damage in rats. Methods A total of 120 clean-grade male Sprague-Dawley (SD) rats were divided into five groups (n = 24) by random number table method, and then given 0 (spontaneous breathing), 10, 20, 30, 40 mL/kg VT for MV. The rats in each group were subdivided into four subgroups of 1, 2, 3, and 4 hours according to ventilation time, with 6 rats in each subgroup. The lung tissue and bronchoalveolar lavage fluid (BALF) were harvested, and alveolar macrophages (AMs) and type Ⅱ alveolar epithelial cells (AECⅡ) were cultured in vitro. The mRNA and protein expressions of autophagy-associated protein microtubule-associated protein 1 light chain 3B -Ⅱ (LC3B -Ⅱ) and autophagy-related genes Beclin1 and p62 were determined by reverse transcription-polymerase chain reaction (RT-PCR) or Western Blot. Lung autophagosome formation was observed under transmission electron microscope. The levels of adenosine triphosphate (ATP), reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in lung tissue were determined for assessing mitochondrial damage. Results There were no significant differences in the mRNA and protein expressions of LC3B -Ⅱ, p62 and Beclin1 at 1 hour after ventilation among the groups. With the prolonged ventilation time, the mRNA and protein expressions of LC3B -Ⅱ, p62 and Beclin1 in MV groups were increased gradually, peaked at 2-3 hours, and they were increased significantly in 30 mL/kg VT group as compared with those in spontaneous respiration group with statistical significances [ventilation for 2 hours: LC3B -Ⅱ mRNA (2-ΔΔCt) was 2.44±0.24 vs. 1.12±0.04, LC3B -Ⅱ/LC3B -Ⅰ was 1.42±0.16 vs. 0.57±0.03, p62 mRNA (2-ΔΔCt) was 2.96±0.14 vs. 1.14±0.02, Beclin1 mRNA (2-ΔΔCt) was 2.80±0.13 vs. 1.14±0.02; ventilation for 3 hours: p62/β-actin was 1.14±0.15 vs. 0.55±0.04, Beclin1/β-actin was 1.27±0.06 vs. 0.87±0.04, all P < 0.05]. Autophagosomes and autolysosomes were found in AECⅡ after ventilation for 2 hours at 30 mL/kg VT by transmission electron microscopy, but not in AECⅠ. Compared with spontaneous breathing group, ATP synthesis in AMs was significantly decreased at 2 hours of ventilation in 30 mL/kg VT group (A value: 0.82±0.05 vs. 1.00±0.00, P < 0.05), ROS accumulate in AMs and AECⅡ were significantly increased [ROS in AMs: (33.83±4.00)% vs. (6.90±0.62)%, ROS in AECⅡ: (80.68±0.90)% vs. (2.16±0.19)%, both P < 0.05]. With the increase in VT and the prolongation of ventilation time, ATP and ROS levels in AMs and AECⅡ were gradually decreased, the ATP (A value) in AMs at 4 hours of ventilation in 40 mL/kg VT group was 0.41±0.05, the ROS in AMs was (12.95±0.88)%, and the ROS in AECⅡ was (40.43±2.29)%. With the increase in VT and the prolongation of ventilation time, MMP levels were gradually increased, the MMP (green/red fluorescence intensity ratio) in AMs at 2 hours of ventilation in 30 mL/kg VT group was 1.11±0.17, the MMP in AECⅡwas 0.96±0.04, and the MMP (green/red fluorescence intensity ratio) at 4 hours of ventilation in 40 mL/kg VT group was 0.51±0.07 and 0.49±0.06, respectively. Conclusion The MV with high VT could induce autophagy activation and mitochondrial damage in lung tissue of rats, and the longer the ventilation time, the more obvious autophagy in the lung.
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Objective To evaluate the effects of alpha-lipoic acid (α-LA)on autophagy in human skin fibroblasts (HSFs). Methods HSFs at passage 3 - 5 were divided into several groups to be cultured with α-LA at final concentrations of 0, 0.01, 0.05, 0.10, 0.15, 0.20 and 0.50 mmol/L for 4, 12 and 24 hours, respectively. Methyl thiazolyl tetrazolium(MTT)assay was performed to evaluate cellular proliferative activity, monodansylcadaverin(MDC)staining to determine autophagy levels, and Western blot to measure the expression of the microtubule-associated protein 1 light chain-3B(LC3-B). Results After incubation for 24 hours, there was a significant difference in the proliferative activity of HSFs among all the groups (F = 10.41, P 0.05). MDC staining also showed a significant difference in the percentage of autophagosome-positive cells among all the groups after 24-hour incubation (F = 8.03, P 0.05). Western blot revealed that the degree of conversion from LC3-Ⅰ to LC3-Ⅱ(LC3-Ⅱ/LC3-Ⅰratio)was significantly different among all the groups after 24-hour incubation (F = 37.49, P 0.05). Conclusion α-LA may inhibit basal autophagy in HSFs.