Objective To investigate the effect of baicalin on CT26.WT cells of colon cancer in mice, and to discuss the cell death form. Methods CT26.WT cells were divided into four groups including of control group , routine cultured in fresh medium, the baicalin group, added with concentration of 100 μmol·L-1 baicalin, the z-VAD-fmk group, was added with final concentration of 20 μmol·L-1 z-VAD-fmk, and the combination group, added final concentration of 20 μmol·L-1 z-VADfmk,1 h before adding 100 μmol·L-1 baicalin. Then the inhibitory effect of baicalin on cell proliferation and cell viability were detected by CCK-8 method. The changes of nucleus were detected by DAPI staining, the ultrastructure of cells was observed by TEM, and the effect of baicalin on the expression of RIP3 gene and protein in cells was detected by QPCR method and Western blotting. Results Compared with control group, the differences of baicalin group and combination group had statistically significance (P<0.05) . cell death rate for control group was (10.54±0.19) ％ ,for baicalin group was (34.93±0.16) ％ ,for z- VAD group was (11.23±0.59) ％, and combination group was (23.27±1.20) ％ (P<0.01) . Compared with the normal control group, baicalin group showed nuclear concentration and fragmentation. there was obvious nuclear fragmentation in the combination group against baicalin group. The results of electron microscopy showed that the cells of baicalin were necrotic, cell swelling, mitochondria swelling and contents leaking. Baicalin group significantly up - regulated RIP3 mRNA expression (P < 0. 01) and enhanced RIP3 protein expression (P < 0. 05) . Conclusion Baicalin induces the necrosis of ct26. WT cells, and can significantly increase the gene and protein expression of RIP3.

A micro droplet generator based on V-shape linear ultrasonic motor was prepared to produce micro droplets with higher accuracy in the field of biochemistry.The device was composed of a micro droplet generator which was driven by the V-shaped linear ultrasonic motor, a three-dimensional displacement platform based on V-shaped linear ultrasonic motor, and a micro droplet separation unit based on the piezoelectric vibrator.The generating part consisted of an ultrasonic motor, a medical syringe, a silica flexible tube and a self-made micro nozzle based on glass.Utilizing the drive controller to drive the linear ultrasonic motor, the slipway pushes forward the syringe and the micro droplet was attached to the glass nozzle.The natural mode of the rod nozzle was excited by the piezoelectric vibrator.The attached droplet was separated from the tip of the nozzle after overcoming the viscous force.The separated droplet fell in a certain range.And the radius of the spherical droplet was calculated.In the experiment, distilled water was used as the initial liquid to investigate the characteristics of the micro droplets produced by the device.The experimental results showed that the droplet was attached to the tip of the micro nozzle which was formed by distilled water under the linear motor.By the vibration of the separation unit, the attached droplets formed the spherical droplets by overcoming the viscous forces in the tip of the nozzle.The radius of spherical droplets generated by this device was less than 40 μm by measuring the size.

OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.

Objective:To study the feasibility of transplantation of normal rat penile corpus cavernosum and major pelvic ganglion (MPG)into the renal subserous region of a Nu /Nu mouse based on allograft technology.Methods:Penile corpus cavernosum and MPG,harvested from Sprague-Dawley (SD)rats under sterile condition,were transplanted underneath the kidney capsule of Nu /Nu mice through the mi-crosurgery instruments and surgery microscope.The histopathologic changes and cellular proliferation in the transplanted penile corpus cavernosum and MPG were then analyzed at the end of 1week and 4 weeks after transplantation.Histological staining and immunohistochemical staining were used to evaluate the main outcome measures.Results:After 1 week,the tissue morphology of the transplanted corpus caverno-sum underneath the kidney capsule of Nu /Nu mice was consistent with normal penile corpus cavernosum, and blood could be observed in the penis cavernous sinus of the graft;after 4 weeks,the mophorlogy of the tranplanted corpus cavernosum near the kidney was consistent with normal penile corpus cavernosum, while fibrosis was noteworthy in the graft away from the kidney,but blood could still be seen in the penis cavernous sinus.After 1 week,the tissue morphology of the transplanted MPG was consistent with normal MPG,multiple islet-like cell clusters could be seen in the transplanted MPG in the renal subserous re-gion,and angiogenesis could be observed near the kidney;after 4 weeks,a network of blood vessels was clearly visible away from the kidney,and islet-like cell clusters were still clearly observed in the trans-planted MPG.In addition,ki67 positive cells were observed in the transplanted penile corpus cavernosum and MPG after 4 weeks of transplantation,which indicated that there was still cell proliferation activity in the grafts.Conclusion:The transplanted corpus cavernosum and MPG underneath the kidney capsule of Nu /Nu mice could survive at least 4 weeks.Moreover,the inner structure of the transplanted corpus ca-vernosum and MPG was close to the normal tissue.The underlining mechanism may be related to the lo-cal microenvironment underneath the kidney capsule of Nu /Nu mice and the neovascularization in the transplanted grafts.

Objective To investigate the risk factors of chylous leakage after pancreatioduodenectomy so as to find effective measures to prevent this complication.Methods A retrospective analysis was conducted on 230 patients who underwent pancreatioduodenectomy at the First Affiliated Hospital of Zhejiang University from Jun.2012 to Jun.2014.Patients with chylous leakage were identified and a 1 ∶ 2 patients in the study and the control groups were selected.The parameters for matching included tumor volume,vascular invasion,and extent of lymph node dissection.A logistic analysis was performed to identify independent risk factors of chylous leakage.Results 15 (6.5％) patients developed chylous leakage after pancreatioduodenectomy.The average hospital stay after surgery of the study group was 20.8 days,compared to 13.5 days in the control-group (P =0.004).In the study group,chylous leakage rate increased in patients with 14th and 16th group of lymph nodes dissection (80％ vs 36.7％,P =0.006).Logistic analysis showed that 14th and 16th lymph nodes dissection was an independent risk factor of chylous leakage after pancreatioduodenectomy (P ＜ 0.05,OR =6.909,95％ CI 1.593 ～ 29.958).Conclusions Chylous leakage prolonged hospitalization after pancreatioduodenectomy.Dissection of the 14th and 16th lymph node groups was an independent risk factor of chylous leakage after pancreatioduodenectomy.Careful ligation of the gastrocolic vein near the lymphatic trunk and dissection of 14th and 16th group of lymph nodes were effective interventions to reduce postoperative chylous leakage.

Objective To evaluate the effects of pregabalin combined with hydrochloric oxycodone on patients with ma-lignant neuropathic pain (MNP). Methods A total of 66 patients with MNP was divided into control group or treatment group randomly. The patients in control group received only hydrochloric oxycodone, and treatment group were treated with the combina-tion of pregabalin and hydrochloric oxycodone. Numeric rating scale (NRS) score was used to evaluate the analgesic effects. Med-ical outcomes study sleep scale (MOS-SS,Chinese version) was used to evaluate the improvement of sleep disorder. The changes of depression or anxiety were investigated by 17-item Hamilton Depression Rating Scale (HAMD-17) or Hamilton Anxiety Scale (HAMA), respectively. Side effects were accessed by Acute and Subacute Toxicity Grading Criteria of Anticancer Drugs (WHO). Results The pain control rate of treatment group was 87. 1% , which was superior to that of control group (58. 6% ) (P<0. 05). The improvement of sleep interference, and the quality and quantity of sleep in treatment group were also superior to that in control group (P<0. 05). After the treatment, depression and anxiety was attenuated in both groups, and the improvement degree in treatment group was higher than that in control group (P<0. 05). No obvious side effects were found in either groups. Conclusion The combination therapy of pregabalin and hydrochloric oxycodone is the better way to treat MNP.

AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .

Objective To explore the effect of irbesartan on cardiac endothelial-mesenchymal transition (EndMT) in diabetic rats.Methods The model of diabetic rat was induced by intraperitoneal injection with streptozotocin (STZ,35 mg/kg) in spontaneous hypertensive rats (SHR).Diabetic rats were divided into diabetic group and the Irbesartan treated group.The pathological changes were investigated by fluorescence microscope and electron microscope.The EndMT was studied in human aortic endothelial cells (HAEC) exposure to high glucose.The concentration of angiotensin Ⅱ in the supernatant was detected by radioimmunoassay.Immunofluorescence staining was performed to detect the co-localization of CD31 and FSP1.Results The significant myocardial fibrosis was presented in the diabetic group.Endothelial protrusions were prominent feature in myocardial microvascular of diabetic rat compared with the control group rats.Double staining of HAEC showed co-localization of CD31 and FSP1,which was decreased by the treatment of Irbesartan (P ＜ 0.05).When HAEC was exposed to high glucose,it showed some cells acquired spindle-shaped morphology and lost CD31 staining,and FSP1 and α-SMA protein expression levels were markedly upregulated,which attenuated by the treatment of Irbesartan.Conclusion Irbesartan might prevent diabetes from myocardial fibrosis via inhibition of EndMT in diabetic rats.

OBJECTIVE To investigate the molecular mechanisms of resveratrol( Res)in renal interstitial fibrosis(RlF)in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight Spra-gur-Dawley rats were randomly divided into UUO( normal saline,n = 16),UUO with Res treatment (Res,20 mg·kg-1 ,n=16),and sham-operation(sham,n=16)models. The kidneys were excised on the 7th and 14th day. The deposition of collagen fiber in the kidney was detected with HE and Masson staining. The levels of sonic hedgehog(SHH,an inducer of SHH pathway)in kidney tissues were deter-mined by ELlSA. lmmunohistochemical analysis was performed to evaluate the protein expression of SHH signaling-related molecules,including SHH,smoothened(Smo),patched-1(Ptch1),and Gli1, proliferating cell nuclear antigen(PCNA)and matrix component typeⅢ collagen. The mRNA expression levels of Smo,Ptch1 and Gli1 were detected by real-time RT-PCR. RESULTS The degree of RlF observed with HE and Masson staining was obviously increased in UUO kidneys,but decreased in Res-treated kidneys. Enhanced expression levels of typeⅢ collagen and PCNA in UUO rats were suppressed by Res treatment(P﹤0.05). Res administration decreased the expression levels of SHH,Smo,and Gli1 (P﹤0.05),but increased the expression of Ptch1(P﹤0.05),suggesting that Res inhibit the obstruction-induced activation of SHH signaling. CONCLUSION Res can attenuate RlF in UUO rats,and the possi-ble mechanism is that Res down-regulates the activity of SHH signaling and inhibits cellular proliferation, resulting in inhibition of matrix accumulation in renal interstitium of UUO rats.

[ABSTRACT]AIM:ToinvestigatetheeffectofimmunosuppressantFK506onserumglucoseinratsandtoex-plore its mechanism .METHODS: Sprague-Dawley rats ( n =12 ) were randomly divided into drug group and normal group.The rats in drug group were intraperitoneally injected with FK 506 at dose of 1 mg· kg-1 · d-1 and the rats in nor-mal group received saline (1 mL· kg-1 · d-1 , ip) for 14 d.The fasting weight and fasting glucose were regularly meas-ured every 2 d.Visceral fat was isolated from the rats at the end of experiment .The mRNA expression of adiponectin , lep-tin, visfatin, resistin, retinol-binding protein 4 ( RBP4) and peroxisome proliferator-activated receptors γ( PPAR-γ) was determined by real-time fluorescence quantitative PCR .The protein expression of PPAR-γand adiponectin was measured by Western blotting .RESULTS:Compared with normal group , the concentration of fasting blood glucose in model group was significantly increased from the 10th day (P<0.05).At day 14, the fasting blood glucose of the model group increased from (5.10 ±0.62) mmol/L to (7.73 ±0.73) mmol/L.No significant change of blood glucose in normal group between the 10th day and the 14th day [from (4.66 ±0.32) mmol/L to (5.80 ±0.10) mmol/L] was observed.Compared with normal group , the mRNA expression of PPAR-γ, adiponectin and leptin in the adipose tissue of model group was signifi-cantly decreased ( P <0.01 ) , whereas the expression of visfatin , resistin and RBP4 was significantly increased ( P <0.05).Compared with normal group, the expression of PPAR-γand adiponectin in model group was decreased (P <0.01).CONCLUSION:FK506 may decrease the expression of PPAR-γto change the expression of adipocytokines and induce hyperglycemia in rats .