Arginine methylation has been implicated in the signal transduction pathway leading to cell growth. Here we show that a regenerating rat liver following partial hepatectomy exhibited elevated methyltransferase activity as shown by increased methylation of a subset of endogenous proteins in vitro. The 20-kDa protein was shown to be a major cytosolic protein undergoing methylation in regenerating hepatocytes. Methylation of the 20-kDa protein peaked at 1 d following partial hepatectomy, which gradually declined to a basal level within the next 14 d. Likewise, methylation of exogenously added bulk histones followed the similar time kinetics as the 20-kDa protein, reflecting time-dependent changes in methyltransferase activity in regenerating hepatocytes. Presence of exogenously added bulk histone in the in vitro methylation assay resulted in dose-dependent inhibition of methylation of the 20-kDa protein. All the histone subtypes tested, histone 1, 2A, 2B, 3 or 4, were able to inhibit methylation of the 20-kDa protein while addition of cytochrome C, a-lactalbumin, carbonic anhydrase, bovine serum albumin, and g globulin minimally affected methylation of the 20-kDa protein. Since methylation of the 20-kDa protein preceded proliferation of hepatocytes upon partial hepatectomy, it is tempting to speculate that the methylated 20-kDa protein by activated histone-specific methyltransferase may be involved in an early signal critical for liver regeneration.
Animals ; Cytoplasm/*chemistry ; *Hepatectomy ; Histones/metabolism ; Humans ; Liver Regeneration/*physiology ; Methylation ; Methyltransferases/metabolism ; Protein Isoforms/metabolism ; Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/physiology ; Subcellular Fractions/chemistry/metabolism

OBJECTIVETo investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).

METHODSWistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).

RESULTS(1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P<0.01, P<0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P<0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P<0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P<0.05 and P<0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P<0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P<0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P<0.05). The concentration of HSP70 did not show any 0.05).

CONCLUSIONSThe TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.

Animals ; Animals, Newborn ; Benzofurans ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; NF-kappa B ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; enzymology ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription, Genetic ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism