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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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Proteins usually associate with other molecules physically to execute their functions. Identifying these interactions is important for the functional analysis of proteins. Previously, we reported the parallel analysis of translated ORFs (PLATO) to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the "bait" molecules, followed by the deep sequencing analysis of mRNA. However, the sample processing, from extraction of precipitated mRNA to generation of DNA libraries, includes numerous steps, which is tedious and may cause the loss of materials. Barcoded PLATO (PLATO-BC), an improved platform was further developed to test its application for protein interaction discovery. In this report, we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis (IBM). Tripartite motif containing 21 (TRIM21) was identified as a potentially new IBM autoantigen. We also expanded the application of PLATO-BC to identify protein interactions for JQ1, single ubiquitin peptide, and NS5 protein of Zika virus. From PLATO-BC analyses, we identified new protein interactions for these "bait" molecules. We demonstrate that Ewing sarcoma breakpoint region 1 (EWSR1) binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4. RIO kinase 3 (RIOK3), a newly identified ubiquitin-binding protein, is preferentially associated with K63-ubiquitin chain. We also find that Zika NS5 protein interacts with two previously unreported host proteins, par-3 family cell polarity regulator (PARD3) and chromosome 19 open reading frame 53 (C19orf53), whose attenuated expression benefits the replication of Zika virus. These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of "bait" molecules.
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Objective To establishment the model of angiotensin Ⅱ (AngⅡ) inducinged cardiomyocyte hypertrophy in H9c2 myocardial cells, and observinge the the changes of ubiquitin binding protein leveljunction protein ubiquitination level. Methods Myocardial cell isolated and subcultured H9c2 were cultured in vitro andmyocardial cell, they were divided into three groups in different AngⅡ concentration (10-8, 10-7, 10-6 mol/L), with in different hours (0, 6, 12, 24, 48, 72 h), and there is also a control group; measured the total protein contentlevel were measured useby BCA method , measured the number of cardiomyocytes in the same area were measured, and the relative surface area of myocardial cells were calculated. The expression of ubiquitin binding protein cardiomyocyte hypertrophy was time- and concentration-dependently induced by AngⅡ, wheremanifested, as total cellular protein content,relative cell surface area and the expression of ubiquitin binding proteinubiquitinated desmin significantly increasing compared with the control group (P < 0.05), which were most significant in 10-7 mol/L AngⅡtreated group for 48 h. Conclusions Cardiomyocyte hypertrophy model induced by 10-7 mol/L AngⅡ for 48 h , was successfully established , where H9c2 cardiomyocyte hypertrophy was induced by 10-7 mol/L AngⅡ in 48 h, and ubiquitin binding proteinubiquitinated desmin significantly increased in cardiomyocyte hypertrophy.