AIM:To optimize the I?B? mutant(I?B?M)gene derived from human placenta tissue by deleting N-terminal phosphorylation sites of serine 32/36,and to construct and identify its replication-deficient recombinant adenovirus(AdI?B?M).METHODS:The I?B?M gene(203-1 003 bp)was acquired by positional cloning,followed by subcloning it into pShuttle and pGEM-T vectors for further PCR,double digestion,DNA sequencing and homology analysis.Subsequently,the expression unit of pShuttle-I?B?M containing CMV promoter,I?B?M cDNA and poly A signals was inserted into Ad5 vector,after which the resultant recombinant adenovirus AdI?B?M was packaged in 293 cells by cotransfection with lipofectamine.Western blotting analysis and electrophoretic mobility shift assay were utilized to detect the AdI?B?M-mediated expression of I?B?M gene in 293 cells and its suppressive effect on phorbol myristate acetate(PMA)-induced nuclear factor ?B(NF-?B)activation in ECV304 cells,respectively.RESULTS:The relevant nucleotide and amino acid sequence of I?B?M gene was consistent with that of GenBank(accession number M69043).The titer of the prepared AdI?B?M was 4.0?10 12 pfu/L.Moreover,the I?B?M gene was expressed in 293 cells,and potently inhibited the PMA-induced NF-?B activation in ECV304 cells in a dose-dependent manner.CONCLUSION:AdI?B?M is a nonvel vector for both efficient transfer and expression of I?B?M gene as well as specific inhibition of NF-?B activity,providing a promising future for gene therapy of asthma.

AIM: To investigate the mechanism of Hexue Mingmu Tablets(HXMMT)in improving diabetic retinopathy(DR)based on transcriptomics.METHODS: Zebrafish DR models were established by 3-day glucose induction(130 mmol/L)starting at 3 days post-fertilization(dpf). Larvae were randomized into four groups: control group(CG; aquaculture water), model group(MG; 130 mmol/L glucose), low-dose HXMMT treatment group(L-HX; 130 mmol/L glucose +7.5 mg/L HXMMT), and high-dose HXMMT treatment group(H-HX; 130 mmol/L glucose +75 mg/L HXMMT), with a 3-day intervention period until 6 dpf. The area and length of eyes, and body length of zebrafish were observed by stereomicroscopy, retinal morphology was observed by hematoxylin-eosin staining(HE), and retinal vessel diameter was observed under fluorescence microscope. Differentially expressed genes(DEGs)were identified by RNA-sequencing(RNA-seq)technology to further elucidate the molecular mechanism of HXMMT in improving DR in zebrafish, and the sequencing accuracy was validated through quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: HE staining demonstrated that the intervention with HXMMT significantly improved the disordered cell arrangement, widened gaps, and thickened inner nuclear layer(INL)in ganglion cell layer GCL); retinal vascular diameter quantification revealed that the retinal vessel diameter of the MG significantly increased compared with the CG, and it was significantly changed after the intervention of HXMMT, with significant efficacy in the H-HX(P<0.05); transcriptomics profiling identified 1 470 reversed DEGs, predominantly enriched in the AMPK signaling pathway, FoxO signaling pathway, retinal developmental processes, and tight junction regulation. Technical validation confirmed strong correlation between qRT-PCR and RNA-seq data(R2=0.8571, P<0.05).CONCLUSION: HXMMT may improve retinal vascular microcirculation disorders in DR by regulating core targets including vsx1, pde6c, arr3a, plk1, fbp1b, foxo1a, pcna, and cdk1, as well as synergistically modulating processes such as retinal development in camera-type eyes, visual perception, microtubule cytoskeletal organization, tight junctions, and the AMPK signaling pathway, Foxo signaling pathway.