Attenuation of the Activation of NLRP3 Inflammasome in Fibroblast Like Synoviocytes of Rheumatoid Arthritis by Baicalin through Regulating the Let-7i-3p/PI3K/Akt/NF-κB Signaling Axis

[Objectives]To study the effect and mechanism of baicalin on the activation of NLRP3 inflammasome in human fibroblast like synoviocytes of rheumatoid arthritis(HFLS-RA).[Methods]To confirm that baicalin alleviated the activation of NLRP3 inflammasome in HFLS-RA,the expression of NLRP3 before and after baicalin treatment was observed by immunofluorescence.Western blot was used to detect the protein expression of p-PI3K,p-Akt,NF-κB p65,NLRP3,ASC and caspase-1 after baicalin treatment for 48 h,and the contents of IL-1 and IL-18 in the supernatents were detected by ELISA.In order to explore the mechanism of baicalin alleviating the activation of NLRP3 inflammasome,the corresponding relationship between let-7i-3p and PIK3CA was verified by double luciferin and Westen blot analysis.The expression of let-7i-3p and PI3K before and after baicalin intervention was detected by RT-qPCR.let-7i-3p interference was used to verify whether baicalin mitigated the activation of enhanced NLRP3 inflammasome.[Results]Baicalin(50 and 100 mg/L)significantly reduced the activation of NLRP3 inflammasome,inhibited the protein expressions of p-PI3K,p-Akt,NF-κB p65,NLRP3,ASC and caspase-1,and the secretion of IL-1 and IL-18.let-7i-3p and PIK3CA had a targeted correspondence,and baicalin up-regulated the expression of let-7i-3p and down-regulated the expression of PIK3CA.Baicalin attenuated the activation of NLRP3 inflammasome enhanced by let-7i-3p interference.[Conclusions]Baicalin can up-regulate let-7i-3p expression,inhibit PI3K/Akt/NF-κB signal transduction,and thus reduce the activation of NLRP3 inflammasome in HFLS-RA.

mgr-mir-9 implicates Meloidogyne graminicola infection in rice by targeting the effector MgPDI

MicroRNAs (miRNAs),a class of small non-coding RNAs,are crucial endogenous gene regulators in a range of animals,including plant-parasitic nematodes.Meloidogyne graminicola is an obligate sedentary endoparasite of rice and causes significant yield losses.A number of studies focused on the roles of M.graminicola effectors during the parasitic process;however,how nematode miRNAs regulate its effectors needs elucidating.In this research,we analyzed a cluster of M.graminicola miRNAs obtained at the second-stage juveniles (J2s) stage that are closely linked to the regulation of M.graminicola effectors.There are 49 767 105 total clean reads obtained from three libraries.A total of 233 known miRNAs and 21 novel miRNAs were identified.Among the known miRNAs,mgr-lin-4,mgr-mir-1,mgr-mir-100,mgrmir-86,mgr-mir-279,mgr-mir-87,mgr-mir-71,mgr-mir-9,mgr-mir-50,mgr-mir-72,and mgr-mir-34 are the most abundant11 miRNAs families.Moreover,the expression levels of selected miRNAs were validated by real-time quantitative PCR.We hypothesized that these miRNAs might regulate the expression of secreted effectors during the J2s stage to facilitate its infection.Consistent with this,we found that mgr-mir-9 targets MgPDI,an important M.graminicola effector mRNA.In addition to that,J2s treated with mgr-mir-9 mimics showed down-regulation of MgPDI expression and reduced reproductive ability,alluding mgr-mir-9 is involved in nematode infection.These results provide novel insight into the regulatory functions of M.graminicola miRNAs during the infection and identify miRNAs and their effector targets as potential key management targets to limit parasite survival during the early stages of infection.

Cloning and characterization of an apolipoprotein C2 promoter in the mouse central nervous system

Apolipoprotein C2 is an important member of the apolipoprotein C family, and is a potent activator of lipoprotein lipase. In the central nervous system, apolipoprotein C2 plays an important role in the catabolism of triglyceride-rich lipoproteins. Studies into the exact regulatory mechanism of mouse apolipoprotein C2 expression have not been reported. In this study, seven luciferase expression vectors, which contained potential mouse apolipoprotein C2 gene promoters, were constructed and co-transfected with pRL-TK into HEK293T cells to investigate apolipoprotein C2 promoter activity. Luciferase assays indicated that the apolipoprotein C2 promoter region was mainly located in the ＋104 bp to ＋470 bp region. The activity of the different lengths of apolipoprotein C2 promoter region varied. This staggered negative-positive-negative arrangement indicates the complex regulation of apolipoprotein C2 expression and provides important clues for elucidating the regulatory mechanism of apolipoprotein C2 gene transcription.

Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

A reporter gene system for screening inhibitors of Wnt signaling pathway

Abnormal activation of canonical Wnt signaling has been associated with various types of cancer.Inhibitory reagents targeting the Wnt signaling have great potential to inhibit the growth of relevant tumors.Here we generated a cell-based screening strategy for identification of antagonists of the Wnt/β-catenin signaling pathway.Stable expression wnt3a was generated in HEK293 cell line,which harbors dual-luciferase reporters.The Wnt signaling in the stably transfected cell line was proved to be very sensitive to(-)-epigallocatechin-3-gallate(EGCG)and lithium chloride(LiCl)treatment,respectively.Natural compounds were screened and a couple of novel inhibitory modulators of the Wnt signaling pathway were obtained.