OBJECTIVE:To establish a method for the simultaneous determination of narirutin,naringin,hesperiden and neo-hesperiden in Weisu granule. METHODS:UPLC was performed on the column of ACQUITY UPLC BEH C18 with mobile phase of acetonitrile-0.2% Phosphoric acid aqueous(gradient elution)at a flow rate of 0.40 ml/min,the detection wavelength was 284 nm, the column temperature was 30 ℃,and the injection volume was 1 μl. RESULTS:The linear range was 9.38-93.75 μg/ml for narirutin(r=0.999 7), 32.25-322.50 μg/ml for naringin(r=0.999 7), 11.25-112.50 μg/ml for hesperiden(r=0.999 9) and 11.88-118.75 μg/ml for neohesperidin(r=0.999 8);limits of quantitation were 20 ng,18 ng,18 ng and 18 ng,the limits of detec-tion were 6 ng,5 ng ,5 ng and 5 ng,respectively;RSDs of precision,stability and reproducibility tests were lower than 2.0%;re-coveries were 96.24%-103.12%(RSD=2.45%,n=6),98.43%-102.10%(RSD=1.42%,n=6),96.10%-101.41%(RSD=2.07%,n=6)and 95.57%-99.06%(RSD=1.44%,n=6),respectively. CONCLUSIONS:The method is rapid and efficient,and suitable for the simultaneous determination of narirutin,naringin,hesperiden and neohesperiden in Weisu granule.

Objective: To research the application of perspective imaging of intense laser proton beam in tumor cells, experi-ments are carried out on an ultra-intense picosecond pulsed laser facility, and the results of perspective imaging on biological samples by high energy proton beam, which is excitated among the interaction of picosecond laser with solid target, is showed. Methods: The laser energy is 12 J, the pulse width of laser is 1.5 ps, the laser focal spot diameter is 15 μm, the coefficientf or the off-axis paraboloid lens is 1.5. The target used in the experiment is made by coating a CH film with 1 μm on the back of the 15 pan-thick Cu. The distance between the proton source (laser spot) and the ant sample is 7 mm (i.e. the object distance), the distance between the sample and the RC film is 63 mm (i.e. the image distance). The detector is a stack of RCF or CR-39 films. Results: The ant is taken as the sample, a high energy proton beam, which is excitated as above on the rear surface of target, makes a 2-dimensional perspective imaging of an ant with high contrast, nanometer spatial resolution and density reso-lution. Conclusions: The result provided possibility of the application of perspective imaging of intense laser proton beam in tumor cells.

Objective To investigate the expression and distribution of Cdc42-interacting protein 4 (CIP4) in renal fibrotic tissue,and the interaction between CIP4 and β-catenin in transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) model of HK-2 cell line.Methods In vivo,the model of renal fibrosis was induced by 5/6 subtotal nephrectomy in rat.Masson staining was used to evaluate the level of renal tissue fibrosis.The expression and distribution of CIP4 was detected by immunohistochemistry.In vitro,the EMT model of HK-2 cell line was induced by TGF-β1 (10 μg/L) for 72 h.Western blotting was used to observe the expression of E-cadherin,β-catenin,CIP4 and α-SMA.Colocalization and interaction of CIP4 and β-catenin were detected by immunofluorescence and immunoprecipitation respectively.Results Compared to sham group,CIP4 expression was increased in group of 5/6 subtotal nephrectomy,CIP4 was mainly distributed in basolateral side of renal tubular epithelia.In vitro,expressions of α-SMA and CIP4 were increased in HK-2 cells stimulated by TGF-β1 for 72 h (2.5and 1.8 folds,respectively) (all P＜0.05),expression of E-cadherin was decreased(P＜0.05).Partial colocalization between CIP4 and β-catenin was detected by immunofluorescence.In control group,CIP4 and β-catenin partially colocalized at the cell membrane.Mter the stimulation of TGF-β1,translocation to nucleus of CIP4 and β-catenin were increased,and partially colocalized in nucleus.The interaction between CIP4 and β-catenin was observed by immunoprecipitation in both control and TGF-β1 stimulated groups.Conclusions Expression of CIP4 in renal fibrotic tissue is increased,which is mainly distributed in basolateral side of renal tubular epithelia.CIP4 and βcatenin partially colocalize and interact with each other.CIP4 may play a role in EMT process through the interaction with β-catenin.

Objective To investigate the expression of Erbin in renal interstitial fibrosis (RIF) and the effect of over-expression of Erbin on transforming growth factor β1 (TGF-(β1)-induced epithelial-mesenchymal transition (EMT) in NRK52E cells. Methods In vivo, the model of renal fibrosis was induced by 5/6 subtotal nephrectomy in rat. Scr and BUN was detected and Masson staining was used to evaluate the level of renal tissue fibrosis. The location and expression of Erbin in renal tissue were detected by immunohistochemistry and Western blotting. In vitro, after NRK52E cells were treated by TGF-β1 (10 μg/L) for 72 h, immunofluorescence and Western blotting were used to obverse the expression and distribution of E-cadherin and α-SMA. The expression of Erbin mRNA and protein were detected by RT-PCR and Western blotting respectively. NRK52E cells were transiently transfected with Prk5-myc-Erbin plasmid via lipofectamine 2000, then the expressions of Erbin, E-cadherin and α-SMA were detected by Western blotting. Results (l)Compared to sham group with Scr (33.96±7.28) μmol/L and BUN (8.11±2.55) mmol/L, rats in 5/6 nephrectomy model with Scr (140.52±61.11) μmol/L and BUN (34.23±7.66) mmol/L revealed renal dysfunction. Masson staining indicated kidney interstitial fibrosis, and the expression of Erbin was significantly increased in renal tissue(2.9 folds), especially in tubular epithelia. (2)In vitro, the expressions of Erbin and α-SMA were markedly increased (2.3 folds and 2.1 folds, P<0.05, respectively) and the expression of E-cadherin was dramatically decreased in NRK52E cells stimulated by TGF-β1, which were consistent with immunofluorescence results. TGF-β1-induced E-cadherin suppression and a-SMA induction could be efficiently blocked by over-expression of Erbin (all P <0.05). Conclusions Erbin is up-regulated in renal interstitial fibrosis, and over-expression of Erbin can partly inhibit renal EMT induced by TGF-β1, which indicates Erbin playing an protective role in renal fibrosis.

Objective To observe the effect of CIP4(Cdc42 interacting protein 4)on human renal tubular epithelial to mesenchymal transition(EMT)induced by transforming growth factor β1(TGF-β1)and to study the associated mechanism. Methods Human proximal tubular epithelial cells (HK-2 cell line) were cultured with TGF-β1 (10μg/L) for 72 hours. The protein expressions of E-cadherin and α-SMA were measured by Western blotting. One set of siRNA oligos specific for CIP4 and CIP4 construction of the entire coding sequence were designed based on the full CIP4 sequence in GenBank. Then HK-2 cells were transfected with CIP4-siRNA or pcDNA3.1-hCIP4 via lipofactamine 2000. The protein expressions of CIP4, E-cadherin and α-SMA were evaluated respectively in control cells, TGF-β1 treated cells, siRNA transfected cells, pcDNA3.1-hCIP4-transfected cells by Western blotting. The distribution of E-cadherin and α-SMA was observed by confocal microscope. After TGF-β1-treated HK-2 cells were interferenced with specific inhibitor of PI3K-Akt (wortmannin) 1μmol/L for 48 hours, Western blotting was used to detect the CIP4 protein in control cells and interferenced cells. Results With TGF-β1 stimulation, the expression of E-cadherin protein was decreased markedly (P＜0.05), and in contract, the expression of α-SMA were increased notably (P＜0.05), which revealed that TGF-β1 could induce EMT. After transfected with CIP4-siRNA, the protein expression of E-cadherin was increased (P＜0.05), and the protein expression of α-SMA was decreased (P＜0.05). The EMT induced by TGF-β1 was effectively reversed. After transfected with pcDNA3.1-hCIP4, the expression of E-cadherin protein was down-regnlated (P＜0.05), and the expression of α-SMA protein was up-regulated compared with control group (P＜0.05), leading to EMT. After HK-2 cells were interferenced with wortmannin for 48 hours, the expression of CIP4 was decreased (P＜0.05). Conclusion TGF-β1 upregulates the expression of CIP4 via PI3K-Akt pathway, and CIP4 may participate in EMT induced by TGF-β1.

Objective To observe the expression and localization of CIP4 (Cdc42 interacting protein-4) in the renal fibrosis and the effect of CIP4 on the expression of E-cadherin,vimentin and β-catenin tyrosine phosphorylation. Methods In vitro, the human tubular epithelial cells (HK-2 cell line) were cultured with 10 μg / L TGF-β1 for 72 h. The protein expressions of CIP4, E-cadherin, vimentin and β-catenin tyrosine phosphorylation were measured by Western blotting; the expression of CIP4 mRNA was detected by RT-PCR. The intracellular distribution of CIP4 was observe by confocal microscope. In vivo, Masson staining was used to evaluate the level of renal fibrosis; the expression and distribution of CIP4 in renal tissue were detected by immunohistochemistry. HK-2 cells were transfected with pcDNA3. 1-CIP via lipofectamine 2000. The expressions of E-cadherin, vimentin and β-catenin tyrosine phosphorylation level in the transfected cells were detected by Western blotting. Results The expressions of CIP4 mRNA and protein were up-regulated in renal tubular EMT cells. Most of CIP4 protein localized in cell membrane, and some was in cytoplasm. After stimulation by TGF-β1, the expression of CIP4 protein both in cytoplasm and nucleus was greatly increased (P ＜0.05),especially in cytoplasm. In vivo, CIP4 was expressed in renal tubular epithelia, but little expressed in glomeruli. In renal from 5/6 nephrectomized rats, CIP4 expression was significantly increased. In the CIP4 transfectants, the expression of CIP4, vimentin and β-catenin tyrosine phosphorylation level were up-regulated (P ＜0.05), but E-cadherin expression was suppressed (P ＜0.05).Conclusion The overexpression of CIP4 is likely to take part in the epithelial-to-mesenchymal transition process, thereby promoting the renal fibrosis.