This study focused on the potential therapeutic effect of baicalin on collagen-induced arthritis (CIA) in rats and the underlying mechanisms. The CIA rats were injected with baicalin (50, 100, or 200 mg/kg) once daily for 30 days. The rats were monitored for clinical severity of arthritis, and joint tissues were used for radiographic assessment and histologic examination. We quantified tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) in experimental animals and used Western blots to assess levels of protein abundance, phosphorylation, and acetylation of nuclear factor (NF)-kB p65 and sirtuin 1 (sirt1) protein expression in joint tissues. Human fibroblast-like synoviocytes from rheumatoid arthritis (HFLS-RA) were adopted in further mechanistic investigations. Baicalin intraperitoneal injection for 30 days dose-dependently blocked clinical manifestations of CIA, such as functional impairment and swollen red paws. Meanwhile, it alleviated collageninduced joint inflammation injury and inhibited the secretion of TNF-a and IL-1b in both rat synovium and HFLS-RA. Further mechanistic investigations revealed that baicalin suppresses NF-kB p65 protein expression and phosphorylation in synovial tissue and human-derived synoviocytes. Moreover, the acetylation of NF-kB p65 was downregulated by baicalin, which negatively correlates with the baicalin-induced upregulation of sirt1 expression in the same conditions. The data indicate that CIA in rats can be alleviated by baicalin treatment via relieving joint inflammation, which is related to the suppression of synovial NF-kB p65 protein expression and the elevation of its deacetylation by sirt1.

RNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of Hibiscus cannabinus infected by Hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmoviridae. After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex in vitro. When the full-length RNAs of Potato virus X, Tobacco mosaic virus, Odontoglossum ringspot virus and Cucumber mosaic virus were used as templates, no detectable RNA was synthesized. Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 39-terminal two C residues at the 39 end in vitro and in vivo. The CCC-39 terminal nucleotide sequence was optimal and nucleotide variations from CCC-39 diminished minus-strand synthesis. In addition, two putative stem-loops (SLs) located within the 39-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis. Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis. HCRSV RNA in which the two SLs were replaced with the SLs of Turnip crinkle virus could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV. Taken together, the specific sequence CCC at the 39 terminus and the two SLs structures located in the 39UTR are essential for efficient minus-strand synthesis of HCRSV.

Sequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of TRC at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.