[Effects of FRNK on activation and migration of hepatic stellate cells].

Objective: To investigate the effect of focal adhesion kinase related non kinase (FRNK) on the activation and migration of hepatic stellate cells (HSCs). Methods: Human liver tissue was divided into healthy control group and fibrosis group from March 2019 to September 2019 in Affiliated Hospital of Guizhou Medical University. C57BL/6 mice were divided into wild type (WT) and FRNK gene knockout type (FRNK-/-) groups. The liver fibrosis model was established with carbon tetrachloride (CCl4). After that, FRNK gene overexpression (Ad-FRNK) was constructed with adenovirus vector. HE and Masson staining were used to evaluate the pathological changes and fiber deposition of liver tissue. Western blot was used to detect the expression of PY397-FAK and α-SMA protein. Mouse primary HSCs were extracted, and the effect of FRNK on HSCs migration was detected by wound healing, activation of Rac and Rho was detected by Western blot. Results: The expression of PY397-FAK protein in human liver tissue with hepatic fibrosis was significantly higher than that in healthy control group (0.88±0.09 vs. 0.73±0.09). FRNK was significantly lower than that in control group(0.68±0.09 vs. 0.79±0.11). After animal model was set up, the degree of liver fibrosis in FRNK-/-mice (153±13)% was more serious than that in WT (100%) group. The expression of PY397-FAK and α-SMA protein was significantly elevated (2.50±0.23 vs. 0.75±0.09, 1.46±0.20 vs. 0.92±0.10). After FRNK gene was re-expressed (100%), the degree of liver fibrosis was mainly reversed [(74±6)%], and the expression of PY397-FAK and α-SMA was accordingly decreased(0.68±0.11 vs. 1.12±0.19,0.68±0.10 vs. 0.85±0.06). In vitro, FRNK inhibited the migration of HSCs [WT∶FRNK-/-∶Ad-FRNK,(339±49)%∶(580±53)%∶(259±33)%] and the activation of Rac and Rho proteins (Rac: 0.54±0.07 vs. 0.91±0.10 vs. 0.77±0.12,Rho:0.45±0.05 vs. 0.64±0.06 vs. 0.53±0.07), all P<0.01. Conclusions: FRNK can inhibit the activation and migration of HSCs which contributed to liver fibrosis. The potential mechanism is related to down regulation of PY397-FAK and inhibition of Rac and Rho activation.

Cellular mechanism of Tß4 intervention in liver fibrosis by regulating NF-κB signaling pathway.

OBJECTIVE: To investigate the inhibitory effect of thymosin-ß4 (Tß4) on the activation of the human hepatic stellate cell line (HSC-LX2) induced by interleukin (IL)-1ß. MATERIALS AND METHODS: There were 5 groups in this study, i.e., blank control group, negative control group (SI-NC, empty plasmid), model group (20 ng/ml of IL-1ß), siRNA-Tß4 knockdown group (IL-1ß and si-Tß4) and Tß4 treatment group (IL-1ß and 1000 ng/ml of Tß4). Cell proliferation rate was measured using the Cell Counting Kit-8 (CCK-8) method. The cell cycle change and percentage of apoptotic cells were determined by Propidium Iodide (PI) DNA staining and Annexin V-fluorescein isothiocyanate (FITC) double staining. Cellular nucleic acid levels of p-IKB and nuclear factor-kappa B (NF-κB)/p65 proteins were measured by fluorescent quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Double immunofluorescence staining and Western blot were used to detect nuclear translocation of NF-κB and p65 and levels of cytoplasmic p-IKB protein and nuclear p65 protein. RESULTS: Due to the G0/G1 phase arrest, the number of cells in the Tß4 treatment group increased, compared with the model group and the siRNA-Tß4 knockdown group (p<0.01). In the same between-group comparison, apoptotic rate in the Tß4 treatment group increased significantly (p<0.05). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were markedly higher in the model group and the siRNA-Tß4 knockdown group than in the blank control group (p<0.01). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were remarkably lower in the Tß4 treatment group than in the siRNA-Tß4 knockdown group (p<0.01). The expression levels of NF-κB/p65 and NF-κB/p50 were significantly lower in the Tß4 treatment group. The expression levels of cytoplasmic p-IKB and nuclear NF-κB/p65 were lower in the Tß4 treatment group than in the model group (p<0.01). CONCLUSIONS: Tß4 significantly inhibited IL-1ß-induced HSC-LX2 cell proliferation. The mechanism may involve decreased activation of the NF-κB pathway, decreased expression of p-IKB and nuclear translocation of p65. Therefore, Tß4 had the effect of reversing liver fibrosis.

Predominance of Cryptococcus neoformans var. grubii multilocus sequence type 5 and emergence of isolates with non-wild-type minimum inhibitory concentrations to fluconazole: a multi-centre study in China.

There are few data on the molecular epidemiology of cryptococcosis in China. Here we investigated the species distribution, molecular types and antifungal susceptibilities of 312 Cryptococcus neoformans species complex isolates from ten hospitals over 5 years. Isolates were identified by internal transcribed spacer (ITS) sequencing and by two matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Multilocus sequence typing (MLST) was used to verify species/variety and to designate molecular types. Susceptibility to six antifungal drugs was determined by the Sensititre YeastOne™ method. Cryptococcus neoformans was the predominant species (305/312 isolates (97.8%), all were ITS type 1, serotype A), of which 89.2% (272/305) were C. neoformans var. grubii MLST sequence type (ST) 5 and 6.2% (19/305) were ST31. Other C. neoformans var. grubii STs were rare but included six novel STs. Only two strains were C. neoformans var. neoformans (both serotype AD). Cryptococcus gattii was uncommon (n = 7, four ITS types) and comprised five MLST STs including one novel ST. For C. neoformans var. grubii, the proportion of isolates with non-wild-type MICs to fluconazole significantly rose in the fourth study year (from 0% (0/56 isolates) in the first year to 23.9% (17/71) in the fourth year), including five isolates with fluconazole MICs of ≥32 mg/L. The study has provided useful data on the species epidemiology and their genetic diversity and antifungal susceptibility. The proportional increase in isolates with non-wild-type MICs to fluconazole is noted.

Clinical significance of anti-glomerular basement membrane antibodies in a cohort of Chinese patients with lupus nephritis.

OBJECTIVES: To evaluate the clinical and laboratory features of patients with lupus nephritis (LN) in the presence and absence of anti-glomerular basement membrane antibodies (anti-GBM) and to establish whether the characteristics of the disease correlate with anti-GBM. METHODS: We performed a retrospective study of 157 hospitalised patients with systemic lupus erythematosus (SLE), 91 of whom had LN. The test for anti-GBM used an enzyme-linked immunosorbent assay (ELISA). Clinical and laboratory data were collected and assessed in LN patients with and without anti-GBM. RESULTS: Anti-GBM was detected in 14 (8.9%) of 157 patients with SLE. All of the 14 patients developed LN; of these, 10 reached the criteria for crescentic glomerulonephritis (CGN) and five were diagnosed as Goodpasture's disease. Serum anti-GBM levels were correlated with the presence of both anti-double-stranded DNA antibodies (anti-dsDNA) and anti-nucleosome antibodies (anti-NuA). Significant differences in extrarenal clinical manifestations were found between anti-GBM-positive and -negative LN patients, with regard to pleuritis, pulmonary haemorrhage, sinusitis, and anaemia in particular. CONCLUSIONS: LN with anti-GBM is not rare in Chinese patients. Anti-GBM, together with the additional nephritogenic potential of anti-dsDNA and anti-NuA, may play an essential role in the pathogenesis of the anti-GBM disease in LN. Therefore, in addition to routine anti-GBM assay, anti-dsDNA and anti-NuA measurements should be performed early to ensure a prompt diagnosis and immediate treatment in patients with anti-GBM-mediated disease.