[Cathepsin B Affects the Activation of Nucleotide-binding Domain and Leucine-rich-repeat-containing Family and Pyrin Domain-containing 3 Inflammasome via Transient Receptor Potential Mucolipin-1].

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(H2O2),calcium-sensitive receptor agonist gadolinium trichloride(GdCl3),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of H2O2.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without H2O2.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with H2O2,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl3(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1ß(P= 0.036)were significantly reduced.Cells in the H2O2 group and H2O2+GdCl3 group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 µmol/L of H2O2 concentration were similar.H2O2-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of H2O2.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the H2O2 +siRNA treatment group and the H2O2 treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.

Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome.

Activated nucleotide binding to the oligonucleotide receptor protein 3 (NLRP3) inflammasome is possibly involved in the pathogenesis of Alzheimer's disease through oxidative stress and neurogenic inflammation. Low expression of the signal transducer and activator of transcription 3 (STAT3) gene may promote the occurrence of neurodegenerative diseases to some extent. To clarify the roles of the NLRP3 inflammasome and STAT3 expression in oxidative stress, (1) SHSY5Y cells were incubated with 1 mM H2O2 to induce oxidative stress injury, and the expression of human-cell-specific signal transduction, STAT3-shRNA silencing signal transduction and STAT3 were detected. Cells were pretreated with Ca2+ chelator BAPATA-AM (0.1 mM) for 30 minutes as a control. (2) Western blot assay was used to analyze the expression of caspase-1, NLRP3, signal transduction and STAT3. Enzyme-linked immunosorbent assay was used to analyze interleukin-1ß levels. Flow cytometry was carried out to calculate the number of apoptotic cells. We found that H2O2 treatment activated NLRP3 inflammasomes and decreased phosphorylation of signal transduction and STAT3 serine 727. BAPTA-AM pretreatment abolished the H2O2-induced activation of NLRP3 inflammasomes, caspase-1 expression, interleukin-1ß expression and apoptosis in SHSY5Y cells, and had no effect in cells with downregulated STAT3 expression by RNAi. The findings suggest that downregulation of signal transduction and STAT3 expression may enhance the oxidative stress mediated by NLRP3, which may not depend on the Ca2+ signaling pathway.

[Quality characterization analysis of single decoction and merger decoction of Baihu and Guizhi].

To study the differences and similarities in pharmaceutical characterization and pharmacodynamic characterization between the single decoction and merger decoction of Baihu and Guizhi. The same technology parameters were used to prepare Baihu and Guizhi single decoticon and merger decoction extracts, and then the differences and similarities in pharmaceutical characterization were analyzed based on their HPLC fingerprint, content of index components, and the extraction content. The pharmacodynamic differences and similarities were analyzed by inflammatory model and pain model. There was no significant difference in HPLC chromatographic peak, but the peak area value reflected the difference of quantity to some extent. It was found that the peak value of single Rhizoma anemarrhenae and Cassia twig decoction was less than the peak of their merger decoction, but the peak value of single honey-fried Licorice root decoction was greater than the peak of merger decoction． The contents of neomangiferin, mangiferin and timosaponin B Ⅱ among index components as well as extraction content in merger decoction were higher than those in single decoction． The contents of liquiritin and glycyrrhizic acid as well as extraction content in merger decoction were lower than those in single decoction． There was no significant difference in the content of cinnamicacid and its extraction content between merger decoction and single decoction. According to the efficacy experiment, both of them showed significant anti-inflammatory and analgesic effects. However, the merger decoction showed faster anti-inflammation effect, and longer analgesic effect. It can be concluded that the merger decoction and single decoction of Baihu and Guizhi have the same material basis, and the merger decoction is better for the dissolution of the active ingredients in this recipe, and is more beneficial to the therapeutic effect.