Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

Zhizhu Decoction Alleviates Intestinal Barrier Damage via Regulating SIRT1/FoxO1 Signaling Pathway in Slow Transit Constipation Model Mice

Objective:To explore the possible effects and mechanism of Zhizhu Decoction(ZZD)on the pathophysiology of slow transit constipation(STC).Methods A total of 54 C57BL/6 mice was randomly divided into the following 6 groups by a random number table,including control,STC model(model),positive control,and low-,medium-and high-doses ZZD treatment groups(5,10,20 g/kg,namely L,M-,and H-ZZD,respectively),9 mice in each group.Following 2-week treatment,intestinal transport rate(ITR)and fecal water content were determined,and blood and colon tissue samples were collected.Hematoxylin-eosin and periodic acid-Schiff staining were performed to evaluate the morphology of colon tissues and calculate the number of goblet cells.To determine intestinal permeability,serum levels of lipopolysaccharide(LPS),low-density lipoprotein(LDL)and mannose were measured using enzyme-linked immunosorbent assay(ELISA).Western blot analysis was carried out to detect the expression levels of intestinal tight junction proteins zona-occludens-1(ZO-1),claudin-1,occludin and recombinant mucin 2(MUC2).The mRNA expression levels of inflammatory cytokines including tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,IL-4,IL-10 and IL-22 were determined using reverse transcription-quantitative reverse transcription reaction.Colon indexes of oxidative stress were measured by ELISA,and protein expression levels of colon silent information regulator 1/forkhead box O transcription factor 1(SIRT1/FoxO1)antioxidant signaling pathway were detected by Western blot.Results Compared with the model group,ITR and fecal moisture were significantly enhanced in STC mice in the M-ZZD and H-ZZD groups(P<0.01).Additionally,ZZD treatment notably increased the thickness of mucosal and muscular tissue,elevated the number of goblet cells in the colon of STC mice,reduced the secretion levels of LPS,LDL and mannose,and upregulated ZO-1,claudin-1,occludin and MUC2 expressions in the colon in a dose-dependent manner,compared with the model group(P<0.05 or P<0.01).In addition,ZZD significantly attenuated intestinal inflammation and oxidative stress and activated the SIRT1/FoxO1 signaling pathway(P<0.05 or P<0.01).Conclusion ZZD exhibited beneficial effects on the intestinal system of STC mice and alleviated intestinal inflammation and oxidative stress via activating SIRT1/FoxO1 antioxidant signaling pathway in the colon.