The rate of corneal graft rejection is still high for high-risk keratoplasty although immune suppression drug is routinely used.The role of traditional Chinese medicine in corneal transplantation is concerned gradually.Heijingpaichitang on the prevention and treatment of rats with high-risk corneal allograft rejection needs further study.Objective This study was to investigate the inhibitory effect of heijingpaichitang on high-risk corneal transplantation immune rejection in rats.Methods Sixteen female SD rats were used as the donors and 32female Wistar rats were served as recipients.The high-risk corneal trasplantation models were established by corneal suture in 32 Wistar rats,and then homogeneity variant SD-Wistar corneal transplantation was performed.The recipients were randomized into model control group,cyclosporinc A (CsA)group,heijingpaichitang group and CsA +heijingpaichitang group.CsA,heijingpaichitang and CsA + heijingpaichitang was orally administered 4 days after operation once per day for 15 days,and normal saline solution was used at the same way in the model control group.Ocular anterior segment reaction was examined under the slit lamp and corneal opacification,edema and neovasculation were scored based on Larkin' s criteria.Rejection index of the corneal graft was recorded and the graft survival time was calculated.The pathological examination of the corneal graft was carried out in all rats,and the inflammatory cells in the corneas and CD4+ cells in the periphery blood were assayed using flow cytometry.The use of the animals complied with ARVO Statement.Results Corneal graft rejection occurred in 10 days after operation in the model control group,12-13 days in the CsA group and heijingpaichitang group and 22 days in the CsA +heijingpaichitang group.Compared with model control group,the scores of the corneal opacification,corneal edema and neovascularization were significantly lower in the CsA group,heijingpaichitang group and CsA+heijingpaichitang group (P＜0.05),and all the scores were declined in the CsA+ heijingpaichitang group compared with CsA group and heijingpaichitang group(P＜0.01),but no significant differences were seen in the scores between the CsA group and heijingpaichitang group(P＞0.05).The mean survival time of grafts was (10.38 ±1.69)days in the model control group,(22.50 ± 3.07) days in the CsA + heijingpaichitang group,with the significant difference (t =-9.790,P =0.000).The pathological examination of graft showed that the lymphocytes and new blood vessels were less in the CsA+heijingpaichitang group compared with CsA group and heijingpaichitang group 15 days after operation.Flow cytometry verified that the number of lymphocytes in graft,CD4+cells and CD4+/CD8+ in periphery blood were significantly lower in the heijingpaichitang group,CsA group and CsA+heijingpaichitang group compared with model control group (P＜0.05).Conclusions Heijingpaichitang can inhibit immune rejection to certain extent in high-risk corneal transplantation rat and has a similar effect to 0.1％ CsA.Heijingpaichitang and 0.1％ CsA have a synergistic effect.

OBJECTIVETo investigate the effect of the RNAi and the chemotherapy drugs nolatrxed on the expression of thymidylate synthase(TS) and the growth of the colorectal carcinoma LOVO cells.

METHODSThe siRNA was constructed targeting the human TS gene, and then transfected into the human colorectal cancer LOVO cells. RT-PCR and Western blot technique were used to observe the TS gene and protein expression levels, and MTT was used to detect cell proliferation after silencing the TS gene. In addition, siRNA and nolatrxed were applied to the LOVO cells to observe the TS protein expression and cell growth.

RESULTSTS siRNA significantly reduced the expression of TS gene and protein in LOVO cells, and inhibited cell growth. The IC50 value of LOVO cells was (1.46±0.25) μmol/L in TS siRNA combined with nolatrexed group, (6.81±0.31) μmol/L in the negative control group, and (6.47±0.43) μmol/L in the single nolatrexed group. After treatment of TS siRNA combined with nolatrexed on LOVO cells for 36 hours, the apoptosis index was higher than that in single TS siRNA and nolatrexed[(62.12±0.89)% vs.(21.56±0.67)% and(40.51±0.83)%, both P<0.05].

CONCLUSIONTS siRNA can partly suppress the expression of TS gene in LOVO cells, inhibit cell proliferation, promote cell apoptosis and enhance cell sensitivity to apoptosis induced by nolatrexd.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; enzymology ; pathology ; Humans ; Quinazolines ; pharmacology ; RNA Interference ; Thymidylate Synthase ; genetics ; metabolism

BACKGROUNDHedgehog (Hh) signaling plays an important role in both embryonic development and postnatal tissue homeostasis. Aberrant Hh activation results in a large variety of cancers. This study was designed to discover novel modulators in Hh signaling pathway.

METHODSWe performed yeast-two-hybrid screening and immunoprecipitation to identify the interaction of Nedd4 and Smo. To verify whether Nedd4 is involved in the regulation of Hh signaling, we monitored the activation of Gli-luciferase reporter by overexpressing Nedd4 together with Gli-luciferase reporter. In order to examine the role of endogenous Nedd4 in regulating Hh signaling, we used a short hairpin RNA (shRNA) interference strategy to silence the Nedd4 expression, and then perform dual-luciferase reporter assay. Statistical comparisons were performed by Student's t tests.

RESULTSWe showed that Nedd4 binds to Smo in the transfected HEK293 cells. Overexpression of Nedd4 alone did not significantly activate the Gli reporter compared to pcDNA3 control (Nedd4 group: dimethyl sulfoxide (DMSO), relative luciferase unit (RLU) 1.87 ± 0.41). However, Smo agonist (SAG)-stimulated activation of Gli-luciferase reporter was markedly potentiated in Nedd4 transfected cells (Nedd4 group: SAG, RLU 13.49 ± 1.04, P < 0.05), indicating that overexpression of Nedd4 increases Gli luciferase reporter activity and Nedd4-induced activation of Hh signaling is activity dependent. In Nedd4 knockdown NIH 3T3 cells, the luciferase reporter activity was measured basally and after SAG treatment. In scrambled cells, compared to DMSO, SAG could activate reporter activity by (4.16 ± 0.84)-fold. In Nedd4 knockdown cells, the luciferase reporter activation by SAG was significantly inhibited (SAG, RLU 1.72 ± 0.24, P < 0.05); knockdown of Nedd4 did not change the basal activity of luciferase activity (DMSO, RLU 0.86 ± 0.11), suggesting that the loss of Nedd4 expression diminishes Gli-dependent activity in the Hh pathway and the regulation of Nedd4 in the Hh signaling pathway is activity-dependent.

CONCLUSIONNedd4 positively regulates the Hh pathway and provides a potential target for inhibiting Hh signaling in cancer therapy.

Animals ; Endosomal Sorting Complexes Required for Transport ; physiology ; HEK293 Cells ; Hedgehog Proteins ; physiology ; Humans ; Mice ; NIH 3T3 Cells ; Nedd4 Ubiquitin Protein Ligases ; Receptors, G-Protein-Coupled ; physiology ; Signal Transduction ; physiology ; Smoothened Receptor ; Transcription Factors ; physiology ; Ubiquitin-Protein Ligases ; physiology ; Zinc Finger Protein GLI1

This study is to investigate the effect and mechanism of puerarin on DNA damage of HaCaT cells induced by UVB. Puerarin pre-treated cells were irradiated with UVB at 30 mJ x cm(-2). Twenty four hours after irradiation, DNA damage was detected by comet assay, ceramide was measured by thin layer chromatography and gas chromatography, intracellular free calcium ion was analyzed by flow cytometry, the phosphorylation level of p38 protein was examined by Western blotting method. Levels of DNA damage, ceramide, free calcium ion and p-p38 protein were elevated in UVB model cells. Contrary to the model group, all indicators above were reduced in all groups pre-treated by puerarin. Puerarin restrains the ceramide accumulation to block downstream p38 MAPK pathway and calcium ion rising, therefore reduces DNA damage in HaCaT cells induced by UVB.
Calcium ; metabolism ; Cell Line ; Ceramides ; metabolism ; DNA Damage ; drug effects ; radiation effects ; Down-Regulation ; Humans ; Isoflavones ; pharmacology ; Keratinocytes ; cytology ; metabolism ; Phosphorylation ; Signal Transduction ; drug effects ; Ultraviolet Rays ; adverse effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective To investigate the effects of advanced glycation end-products(AGEPs)on the function of normal keratinocytes in vitro so as to explore the role of AGEPs in impaired wound healing. Methods Normal rat keratinocytes were incubated with different concentrations of AGEPs.After 48 hours of culturing,the cell proliferation rates were measured by MTT colorimetric determination.The cell cycle distributions and apoptosis were analyzed with flow cytometry,and the migration was investigated by 24-well fluorimetric cell migration assay kit by exposing to 100?g/ml AGEPs.Nuclear extracts from these cells were examined for binding of nucleotides containing NF-?B consensus by immunocytochemistry and EMSA in vitro.Results The proliferations of normal keratinocytes were significantly arrested and many cells were induced to early apoptosis compared with control ones(P＜0.05)by exposing to AGEPs for 48 hours. Meanwhile AGEPs also irritated keratinocytes migration compared with control ones(P＜0.05).Inhibiting the activation of NF-?B could partly recover the proliferation of keratinocytes,reverse apoptosis and attenu- ate migration.Conclusion AGEPs are correlated with the migration,proliferation and apoptosis of kera- tinocytes by NF-?B.

OBJECTIVETo compare the difference in the efficacy on gait time cycle of ischemic stroke between yin-yang respiratory reinforcing and reducing needling technique (yin-yang needling) and the conventional acupuncture.

METHODSSixty cases of ischemic stroke were randomized into a conventional acupuncture group and a yin-yang needling group, 30 cases in each one. The basic treatment (the control of blood pressure, blood sugar and blood lipid, the intravenous drops of ginkgo leaf extract and dipyridamole injection and vinpocetine injection) were applied in the two groups. Additionally, in the conventional acupuncture group, the acupoints of the Stomach Meridian of Foot-Yangming [Biguan (ST 31), Liangqiu (ST 34), Zusanli (ST 36), Shangjuxu (ST 37), Xiajuxu (ST 39), etc.] were selected and stimulated with the even needling technique. In the yin-yang needling group, the acupoints of yin meridians such as Zuwuli (LR 10), Xuehai (SP 10), Yinlingquan (SP 9) and Sanyinjiao (SP 6), etc. and the acupoints of yang meridians such as Biguan (ST 31), Liangqiu (ST 34) and Yanglingquan (GB 34), etc. were selected. The reducing manipulation of respiratory reinforcing and reducing technique was applied to the acupoints of yin meridians and the reinforcing manipulation was applied to the acupoints of yang meridians. The kinematics time parameters were determined and compared before and 4 weeks after treatment.

RESULTSAfter treatment, the differences in the gait cycle, the phase time of standing (%), the phase time of single support (%), the phase time of unilateral sway (%) on the affected (healthy) foot and phase time of double support (%) were significant as compared with those before treatment in the patients of the two groups (all P < 0.05), in which, the gait cycle (1.75 +/- 0.21 vs 2.02 +/- 0.37), the phase time of standing (%) on the affected (healthy) foot [(65.41 +/- 5.20)% vs (68.37 +/- 6.24)%, (70.99 +/- 6.47)% vs (74.51 +/- 5.19)%], the phase time of unilateral sway (%) on the affected (healthy) foot [(36.08 +/- 4.86)% vs (33.65 +/- 2.94)%, (31.04 +/- 3.41)% vs (26.77 +/- 2.67)%] and the phase time of double support (%) [(36.91 +/- 5.10)% vs (41.22 +/- 5.39)%] in the yin-yang needling group were improved much obviously after treatment as compared with those in the conventional acupuncture group. The differences in support phase time (%), single support phase time (%) and sway phase time (%) were significant between the affected limb and healthy limb of the two groups after treatment (all P < 0.05).

CONCLUSIONYin-yang respiratory reinforcing and reducing needling technique effectively improves hemiplegic gait movement cycle and walking function in patients of ischemic stroke, which is superior to the conventional acupuncture treatment.

Acupuncture Therapy ; instrumentation ; methods ; Aged ; Female ; Gait ; Humans ; Hypoxia-Ischemia, Brain ; physiopathology ; therapy ; Male ; Meridians ; Middle Aged ; Stroke ; physiopathology ; therapy ; Time Factors ; Yin-Yang

OBJECTIVETo investigate the inhibitory effect of lactic acid on semen-derived amyloid (SEVI) fibril formation.

METHODSPAP248-286 (2 mg/mL) was incubated with 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mg/mL of lactic acid. After incubation for different times, aliquots were drawn from each sample for Thioflavin T (ThT) and Congo red staining to monitor semen-derived amyloid fibril formation. The β sheet structure formation of PAP248-286 was measured by circular dichroism spectrum, and the morphology of amyloid fibrils incubated with or without lactic acid was observed with transmission electron microscopy (TEM). The enhancing effect of amyloid fibril incubated with lactic acid at different time points was determined using virus infection assay. PAP248-286 (2 mg/mL) was incubated with dilutions of vaginal secretion from healthy women, and amyloid fibril formation was detected with ThT and Congo red staining.

RESULTSLactic acid inhibited SEVI fibril formation in a dose-dependent manner in vitro. Lactic acid at 0.5 mg/mL completely inhibited 2 mg/mL SEVI fibril formation within 48 h. After incubation for 48 h, lactic acid at 1 mg/mL inhibited the formation of β-sheet structure of SEVI (2 mg/mL) and completely inhibited 2 mg/mL PAP248-286 aggregation as observed with TEM. In the presence of lactic acid, PAP248-286 lost the ability to enhance virus infection. Vaginal secretion inhibited SEVI fibril formation in a dose-dependent manner, and virtually no SEVI fibril occurred after incubation of 2 mg/mL PAP248-286 with 67% vaginal secretion.

CONCLUSIONLactic acid inhibits SEVI fibril formation in vitro.

OBJECTIVETo study the fingerprint of Zedoary Turmeric Oil (ZTO) as the bulk drug of Kingkong Elemene for making it safe, effective, stable, and controllable.

METHODSFingerprints were detected by gas chromatography. β-elemene peak was regarded as reference peak (S). The relative peak area of each common peak and the relative retention time were calculated. With a total of modes for reference, the fingerprints of 10 batches of Kingkong ZTO were detected, and their similarity was calculated by traditional Chinese medicine (TCM) fingerprint similarity calculation software.

RESULTSThe determination method was stable and reliable. Totally 19 common characteristic peaks of Kingkong ZTO was found. The fingerprint similarity of these batches of Kingkong ZTO were not lower than 0.96.

CONCLUSIONSGas chromatography for detecting the fingerprint of Kingkong ZTO was reliable and repeatable. The established fingerprint of Kingkong ZTO could guarantee the quality stability and safety of different product batches.

Chromatography, Gas ; Curcuma ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Plant Oils ; chemistry ; Sesquiterpenes ; chemistry

OBJECTIVETo investigate the effect of pulmonary surfactant (PS) on the Th1/Th2 balance and serum levels of interleukin-4 (IL-4), interferon-γ (IFN-γ) and IgE in neonates with respiratory distress syndrome (RDS).

METHODSA total of 58 neonates with RDS were divided into control (n=20) and PS treatment groups (n=38). The control group underwent mechanical ventilation and other conventional treatment, while the PS treatment group received with bovine PS treatment within 1 hour of being admitted to the hospital together with mechanical ventilation and other conventional treatment. Enzyme-linked immunosorbent assay was used to measure serum levels of IL-4, IFN-γ and IgE before treatment and 24, 48 and 72 hours after treatment. Simultaneously, arterial blood gas, respiratory system compliance, and other ventilator parameters were recorded.

RESULTSCompared with the control group, the PS treatment group showed significantly shorter duration of mechanical ventilation and oxygen exposure time (P<0.05), significantly better respiratory system compliance and significantly lower oxygenation index 24, 48 and 72 hours after treatment (P<0.05). At 48 and 72 hours after treatment, serum levels of IFN-γ were significantly lower in the PS treatment group than in the control group (120±46 ng/L vs 229±59 ng/L, P<0.05; 141±40 ng/L vs 282±44 ng/L, P<0.05), and serum levels of IL-4 were significantly higher in the PS treatment group than in the control group (263±48 pg/mL vs 152±45 pg/mL, P<0.05; 417±49 pg/mL vs 201±46 pg/mL, P<0.05). At 72 hours after treatment, serum level of IgE was significantly lower in the PS treatment group than in the control group (115±44 pg/mL vs 199±43 ng/mL; P<0.05).

CONCLUSIONSPS treatment can shorten the duration of mechanical ventilation and oxygen exposure time, regulate serum levels of IFN-γ, IL-4 and IgE, and influence Th1/Th2 balance in neonates with RDS, thus inhibiting lung inflammatory response and reducing lung injury.

CD4 Lymphocyte Count ; Female ; Humans ; Immunoglobulin E ; blood ; Infant, Newborn ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Male ; Pulmonary Surfactants ; pharmacology ; therapeutic use ; Respiration, Artificial ; Respiratory Distress Syndrome, Newborn ; complications ; drug therapy ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo investigate the effect of augmentative plate fixation to increase stability in the treatment of femoral shaft nonunions subsequent to intramedullary fixation.

METHODSNine patients with femoral nonunions after intramedullary nail internal fixation were treated with augmentative plate internal fixation from April 1998 to Jane 2008, included 8 males and 1 female, with an average age of 32 years old ranging from 21 to 54 years. One case was upper 1/3 femoral fractures, 5 cases were middle 1/3 femoral fractures, 3 cases were lower 1/3 femoral fractures. The interspace of bone nonunion was more than 5 mm in 6 cases, of them, iliac bone grafting were applied in 4 cases, artificial bone combined with iliac bone grafting were applied in 2 cases; The interspace of bone nonunion was less than 5 mm in other 3 cases,artificial bone grafting was applied in 1 case, fitting bone callus were applied in 2 cases. All patients got protected weight loading preventing the main screw break.

RESULTSAll patients achieved radiological solid union at an average of 8 months (ranged 6 to 11 months ). The fixation was removed during 6 to 11 months after operation in 5 cases. Donor site pain of iliac occurrenced on 4 cases,3 cases relieved 1 month later and 1 case relieved 3 months later. No infection, fixation loosening or breaking was observed.

CONCLUSIONThe augmentative plate fixation can be applied at the fracture site to prevent the rotational instability. The technique is simple and does not require any special instrument, which facilitates an early weight bearing and gives a quick recovery from nonunion.

Adult ; Bone Plates ; Female ; Femoral Fractures ; surgery ; Femur ; pathology ; Fracture Fixation, Intramedullary ; methods ; Fractures, Ununited ; surgery ; Humans ; Hypertrophy ; Male ; Middle Aged