The roles of AMPK-mediated autophagy and mitochondrial autophagy in a mouse model of imiquimod-induced psoriasis.

BACKGROUND: Psoriasis is a systemic inflammatory disease characterized by epidermal hyperplasia and skin inflammatory infiltrates. Inactivation of AMPK has been shown to decrease autophagy, thereby inhibiting elimination of inflammatory factors and harmful substances, and aggravating psoriasis. However, the molecular mechanism through which AMPK affects psoriasis remains to be further explored. In this study, we investigated whether AMPK regulates autophagy through the ULK1/Atg7 signaling pathway and regulates mitochondrial autophagy through the PINK1/Parkin signaling pathway, thereby affecting a mouse model of psoriasis. METHODS: Imiquimod was used to induce psoriasis-like lesions on the backs of mice. The severity of skin lesions in psoriatic mice was evaluated with the skin inflammation severity score, and epidermal thickness was measured on the basis of H&E staining. RT-PCR, western blotting and immunofluorescence staining were used to detect indicators of autophagy and mitochondrial autophagy. RESULTS: AMPK activity was inhibited in the psoriasis mouse model, the autophagy-associated proteins ULK1/Atg7 were inhibited, and the mitochondrial autophagy proteins PINK1/Parkin were also decreased. Results indicated that autophagy and mitochondrial autophagy were inhibited in the mouse model. When AMPK signaling was upregulated, ULK1/Atg7 and PINK1/Parkin were upregulated, autophagy and mitochondrial autophagy increased, and skin lesions in the mouse model were alleviated. ULK1/Atg7 and PINK1/Parkin were down-regulated when AMPK signaling was downregulated, and psoriasis-like skin lesions were aggravated in mice. These results indicated that AMPK regulates autophagy through the ULK1/Atg7 signaling pathway and regulates mitochondrial autophagy through the PINK1/Parkin signaling pathway, thus affecting the prognosis of psoriasis in the mouse model. CONCLUSION: AMPK affects the prognosis of psoriasis in a mouse model by regulating autophagy and mitochondrial autophagy.

Interleukin-12 receptor ß2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation.

Interleukin-12 receptor ß2 (IL-12Rß2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rß2 in teleost fish. In this work, we have cloned and characterized IL-12Rß2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rß2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rß2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rß2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rß2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rß2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rß2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rß2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rß2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rß2 is involved in the regulation of intestinal inflammation.

[Comparison of chemical constituents between Cyathula offinalis and adulterant and their admixture by HPLC characteristic chromatogram fingerprint combined with chemometrics].

Cyathula capitate is the main adulterant of C.offinalis. According to the literature reported, there are obvious differences in properties, taste and pharmacological activity between C. capitate and C.offinalis. Therefore, C. capitate can only be used as a local conventional medicine and can't be a substitute for C. offinalis. Since the appearance of C.capitata is very similar to the C.offinalis and the content of cyasterone also can reach the limit of the current pharmacopoeia standard, the C.capitata is mostly sold in the form of impersonation oradmixture, which seriously affected the safety of the clinical medication and the development of the genuine crude drugs. In view of this, HPLC characteristic fingerprint was used to reveal the difference of multi-ingredients of C. offinalis, C. capitata and their admixture. According to the HPLC chromatogram of C.offinalis, C. capitata. and their admixture, 65 different components were obtained to set up a peak area data matrix of 26×65, which was applied to perform the characteristic peak difference analysis, similarity analysis, hierarchical clustering analysis HCA and principal component analysis (PCA). Characteristic peak difference analysis showed that the characteristic peaks of C. capitata and their admixture are more and higher respond than those of C. offinalis. The 9 characteristic peaks were used to distinguish C. capitata, 2 of which were used to distinguish C. offinalis mixed with 5% C. capitata. UV spectra of 9 characteristic peaks are mostly similar to the end absorption spectra of saponins, indicating that C. capitata may contain a large amount of saponins. By the reference fingerprint of C.offinalis established, the similarity analysis showed that the similarity degree of C. offinalis are higher than 0.942, while the similarity degree of C. capitata, C.offinalis mixed with 5% C. capitata are less than 0.383 and 0.399. C.offinalis, C. capitata, C.offinalis mixed with 5% C. capitata could be obviously divided into 3 classes by HCA and PCA. These results showed that there are obvious difference in the chemical composition of C. offinalis, C. capitata and their admixture, which could provide evidence for their identification.

[Construction of the transfected cell line expressing the human CXCR4 gene and study of its biological function].

AIM: To construct the tranfected cell line expressing the human CXCR4 gene and to study the biological function. METHODS: The total RNA was isolated from peripheral blood mononuclear cell (PBMC) with TRIzol, and the CXCR4 gene was amplified by RT-PCR, then digested with restriction endonuclease Pst I and EcoR I, and inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was co-transfected into the package cells 293T with LipofectAMINE 2000. Then the supernatant of the 293T cell culture was used to infect L929 cells, the cell clones stably expressing the CXCR4 molecule were screened in the presence of Zeocin (500 mg/L) after 72 h cultivation. RESULTS: It was found that the full-length of CXCR4 gene was successfully cloned, and the recombinant retrovirus vector carrying the CXCR4 gene was constructed. The CXCR4 cDNA transfected L929 cell could stably express the human CXCR4 on the cell membrane, and the migration ability of transfected cells was well evidenced in the transwell system induced by SDF-1alpha after the transfection with CXCR4. CONCLUSION: The CXCR4 transfected L929 cell line was successfully established, and it can make the basis for the further research.