Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure / 中华肝脏病杂志

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in β-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.