Conjugation of (-)-epigallocatechin-3-gallate and protein isolate from large yellow croaker (Pseudosciaena crocea) roe: improvement of antioxidant activity and structural characteristics.

BACKGROUND: Large yellow croaker (Pseudosciaena crocea) roe is the main by-product in the processing of large yellow croaker. Previous studies have found that its protein isolates are composed of vitellogenin, as well as vitellogenin B and C, having good functional properties. (-)-Epigallocatechin-3-gallate (EGCG) is a natural antioxidant component that can be combined with protein to improve antioxidant activity and structural characteristics of protein. RESULTS: EGCG was bound with the P. crocea roe protein isolate (pcRPI) by the free radical method to prepare the conjugate. The formation of pcRPI-EGCG conjugates was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography, which showed that the calculated weight-average molar masses of native-pcRPI and pcRPI-EGCG conjugates were 86.9 and 215.3 kDa, respectively. The results of fluorescence, ultraviolet, circular and infrared spectra indicated that the conjugation of EGCG with native-pcRPI changed the secondary and tertiary structure of native-pcRPI. The pcRPI-EGCG conjugates exhibited higher thermal stability than native-pcRPI. The radical scavenging and reducing power of native-pcRPI were increased by 2.0-2.5- and 1.4-fold, respectively, after the EGCG-grafting reaction. CONCLUSION: These results indicate that the binding of pcRPI and EGCG effectively improved the antioxidant properties and structural characteristics of the pcRPI. © 2021 Society of Chemical Industry.

Upregulation of cell adhesion through delta Np63 silencing in human 5637 bladder cancer cells.

Some researchs have demonstrated that the loss of delta Np63 is associated with aggressive phenotypes and poor prognosis. However, other research indicates that delta Np63 is considered to have oncogenic properties. Delta Np63 overexpression is often observed in association with the oncogenic growth of squamous cell carcinomas and bladder cancer. In this study, we investigated the oncogenic role of delta Np63 in regulating cell adhesion in transitional cell carcinoma of the bladder (TCCB). The cells were stably transfected with the delta Np63 short hairpin RNA (shRNA) plasmid. Immunocytochemistry was performed to determine the knockdown efficiency. Tumour cells were studied for their ability to adhere to vascular endothelial cells. Confocal microscopy was used to analyse the changes in cytoskeletal F-actin. F-actin expression was measured by flow cytometry. Cell invasion ability was assessed using transwell chambers. The delta Np63-silenced tumour cells were shown to adhere more tightly than controls to vascular endothelial cells (P<0.05). The content of F-actin in the delta Np63-silenced cells was enhanced (P<0.05). The Matrigel invasion assays showed that human 5637 bladder cancer cells had a lower degree of motility when transfected with pdelta Np63-shRNA (P<0.05). In conclusion, silencing of the delta Np63 expression can enhance the adhesiveness of 5637 cells by inducing F-actin cytoskeleton production, and it will possibly inhibit the TCCB invasion and metastasis.

Transforming growth factor-ß1 short hairpin RNA inhibits renal allograft fibrosis.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is known to be a key fibrogenic cytokine in a number of chronic fibrotic diseases, including chronic allograft nephropathy. We examined the effects of inhibition of TGF-ß1 expression by RNA interference on renal allograft fibrosis, and explored the mechanisms responsible for these effects. METHODS: A Sprague-Dawley-to-Wistar rat model of accelerated kidney transplant fibrosis was used. Sixty recipient adult Wistar rats were randomly divided into four groups: group J (sham-operated group), group T (plasmid-transfected group), group H (control plasmid group), and group Y (transplant only group). Rats in group T were transfected with 200 µg of TGF-ß1 short hairpin RNA (shRNA). Reverse transcription-polymerase chain reaction and Western blotting were used to examine the expression of TGF-ß1, Smad3/7, E-cadherin, and type I collagen. The distribution of type I collagen was measured by immunohistochemistry. The pathologic changes and extent of fibrosis were assessed by hematoxylin and eosin and Masson staining. E-cadherin and α-smooth muscle actin immunohistochemical staining were used to label tubular epithelial cells and fibroblasts, respectively. RESULTS: Plasmid transfection significantly inhibited the expression of TGF-ß1, as well as that of its target gene, type I collagen (P < 0.05 and P < 0.01, respectively). In addition, the degree of fibrosis was mild, and its development was delayed in plasmid-transfected rats. In contrast, TGF-ß1-shRNA transfection maintained the expression of E-cadherin in tubular epithelial cells while it inhibited the transformation from epithelial cells to fibroblasts. Blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P < 0.05 and P < 0.01, respectively). CONCLUSIONS: This study suggests that transfection of a TGF-ß1-shRNA plasmid could inhibit the fibrosis of renal allografts. The mechanism may be associated with the downregulation of Smad3 and upregulation of Smad7, resulting in suppressed epithelial-myofibroblast transdifferentiation and extracellular matrix synthesis.

[Effects of silencing transforming growth factor-beta1 by RNAi upon transdifferentiation of renal allograft tubular epithelial cells in rats].

OBJECTIVE: To investigate the effects of shRNA-transforming growth factor (TGF)-beta1 plasmid upon epithelial-myofibroblast transdifferentiation of renal allograft in rats. METHODS: Divided the Wistar rats into 4 groups: Group J (sham-operated group), T (plasmid group), H (vacant plasmid group) and Y (simply transplantation group). The SD to Wistar rat transplant kidney-sclerosis accelerated model was constructed and transfected with the plasmid based on hydromechanics. Transplanted kidneys were collected at Months 1, 2 and 3 post-transplantation. The gene transcriptional levels of TGF-beta1 and E-cadherin were detected by RT-PCR and the protein variation of E-cadherin was examined by Western blotting. The pathological changes and infiltrated inflammatory cells were assessed by HE staining and the immunohistochemical staining of E-cadherin and alpha-SMA used to label epithelial cells and fibroblast in order to exhibit cell transdifferentiation. RESULTS: Compared with Group H and Y, the mRNA transcription of TGF-beta1 was obviously inhibited in the Group T: at Month 3, the TGF-beta1 mRNA of Group T is 0.73 +/- 0.08, significantly lower than Group H and Y (0.92 +/- 0.07 and 0.95 +/- 0.04, both P < 0.01); the expression of E-cadherin was maintained at a high level: at the Month 3, the E-cadherin mRNA of Group T is 0.39 +/- 0.11, significantly higher than Group H and Y (0.15 +/- 0.07, and 0.17 +/- 0.06, both P < 0.01); the E-cadherin protein of Group T is 0.38 +/- 0.08, significantly higher than group H and Y (0.15 +/- 0.07 and 0.15 +/- 0.07, both P < 0.01); epithelial cells were much more and fibroblast was much less than that of Group H and Y; there were also less infiltrated chronic inflammatory cells and extracellular matrix deposition in the Group T. CONCLUSION: The shRNA-TGF-beta1 plasmid can inhibit the epithelial-myofibroblast transdifferentiation of renal allograft in rats. The mechanisms may be associated with its effects of down-regulating TGF-beta1 and up-regulating E-cadherin.