Content determination of six flavonoids in Dendrobium officinale stems from different producing areas,cultivation and processing methods by QAMS combined with dual-wavelength method / 中国中药杂志

A novel HPLC method with the quantitative analysis of multi-components by single marker( QAMS) combined with the dual-wavelength method was developed for simultaneous determination of six flavonoids in Dendrobium officinale stems from different producing areas,cultivation and processing methods to clarify the main factors contributing to the different composition of flavonoids.The separation of six flavonoids was performed on a Shiseido Capcell PAK MGⅡ C18 column( 4. 6 mm×250 mm,5 μm) using a linear gradient elution system of acetonitrile-0. 1% formic acid aqueous solution. Schaftoside,isoschaftoside,vicenin-2,and glucosylvitexin were simultaneously analyzed using rutin as a reference standard at detection wavelength of 340 nm,and naringenin was determined at290 nm. The credibility and feasibility of QAMS method were validated and the results demonstrated that no significant differences were observed as compared with the external standard method. Finally,a total of 82 batches of D. officinale samples were analyzed and principal component analysis( PCA) and discriminant analysis were applied to distinguish and compare D. officinale samples from different producing areas,cultivation and processing methods. The results showed that the total flavonoid content of D. officinale stems cultivated in the simulated wild( attached tree cultivation or attached stone cultivation) was significantly higher than that in greenhouse bed cultivation. The content of flavonoids in simulated-wild D. officinale stems was higher in Jiangxi,Guizhou,Zhejiang,and Fujian provinces,while that in greenhouse bed cultivation was higher in Fujian and Zhejiang provinces. The content of naringenin was positively correlated with processing temperature,and that of the other five flavonoids was negatively correlated with processing temperature. PCA showed that wild-simulated D. officinale and greenhouse bed-cultivated D. officinale could be roughly divided into two clusters. The samples cultivated in the greenhouse bed were divided into four categories according to the geographical habitats. Wild-simulated D. officinale samples from Guizhou gathered together,and there was no obvious rule in samples from other producing areas. The established method simplified the determination method of flavonoids in D. officinale,and could provide the basis for effective quality control,cultivation and processing of D. officinale.

Mouse Model of Cisplatin Induced Ototoxicity / 中山大学学报(医学科学版)

@#【Objective】To establish a reliable and reproducible mouse model of cisplatin induced inner ear hair cells loss and to make possible for further study of mechanisms of cisplatin-induced ototoxicity in mice from the molecular and genetic aspects. 【Methods】Twenty healthy mice were evenly divided into two groups in random. The mice in group A received a single dose of furosemide（200 mg/kg，intraperitoneal）followed by a single dose of cisplatin（1.0 mg/kg，intraperitoneal）1 h later，and the mice in group B received a single dose of furosemide（200 mg/kg，intraperitoneal）followed by a single dose of cisplatin（0.5 mg/kg，intraperitoneal）1 h later. Auditory brainstem response（ABR）and distortion product otoacoustic emission （DPOAE） were tested in both groups 10 d post the cisplatin-furosemide treatment and bilateral cochlea were taken out and fixed，one side cochlea were used for the surface preparation of total cochlear basal membrane and stained to count the number of cochlear hair cells from apex to base，the other was embedded with EPON 812 gel and then cut into half thin slices. Six mice were used as control group. 【Results】In both groups，the average ABR threshold of mice at each frequency were elevated，while the average DPOAE values were decreased. The average ABR threshold in group A increased significantly and the amplitude of each frequency decreased a lot，and DPOAE in high frequencies vanished；In group B，there was a minor elevation of ABR threshold with a slightly lower amplitude in higher- frequencies（16，20，32 kHz）but no amplitude change in lower frequencies（4，8，12 kHz）both in ABR and DPOAE. The surface preparation of Cochlear results in group A showed that nearly all the outer hair cells in the base had been destroyed，and most were destroyed in the apex；In group B，only the majority of cochlear outer hair cells at the base missed. There was no decrease of the numbers of the inner hair cells in both groups. Semi-thin cross-sections results also showed that the stria vascularis and spiral ganglion cells were close to normal in both groups. 【Conclusion】Co- administration of single dose of 1.0 mg/kg cisplatin with a single dose of 200 mg/kg furosemide may induce significant loss of cochlear outer hair cells in mice.

Treatment of ureaplasma urealyticum infection patients of Qi deficiency blood stasis syndrome by pengyan pill: a clinical observation / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the clinical efficacy of penyan pill (PP) in treating ureaplasma urealyticum (UU) infection patients of qi deficiency blood stasis syndrome (QDBSS).</p><p><b>METHODS</b>Totally 188 UU infection patients of QDBSS were randomly assigned to two groups, the treatment group and the control group. Patients in the treatment group were treated with PP (10 g each time, thrice daily, 14 consecutive days as one therapeutic course), while those in the control group took azithromycin (10 g each day, 7 consecutive days as one therapeutic course). They were continually treated for 3 therapeutic courses. The clinical symptom integrals were observed in the two groups before and after treatment. The short-term efficacy was judged. Their recurrence rates were followed-up to assess their long-term efficacies.</p><p><b>RESULTS</b>The total effective rate of the comprehensive efficacy in the treatment group was 91.4%, while it was 79. 3%in the control group, showing no statistical difference between the two groups (P > 0.05). Better effects were obtained in improving Chinese medical clinical symptoms in the treatment group (P <0.01). There was no statistical difference in the negative conversion rate between the two groups after treatment (P >0. 05). There was statistical difference in the recurrence rate between the two groups (12. 82% vs 54.76%,P <0. 05).</p><p><b>CONCLUSIONS</b>PP showed equivalent effects in treating UU infection patients of QDBSS to those of azithromycin. But PP showed obvious advantages over azithromycin in improving Chinese medical syndromes.</p>

Hair cells apoptosis and the activation of P53 protein in intensive impulse noise induced cochlear lesion / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the pattern of hair cell injury and expression of P53 apoptosis protein in intensive impulse noise injured cochlear hair cells in guinea pigs.</p><p><b>METHODS</b>Twelve adult guinea pigs were exposed to a series of 40 pairs of impulse noise (2 second intervals) at the intensity of 168 dB (SPL). Animals were terminated at 3, 6 and 12 hours after noise exposure, respectively. Cochlear surface preparations were performed with a double staining of FITC-conjugated phalloidin and propidium iodide for the observations of the stereocilia and the nucleus. P53 immunochemical staining was also performed 12 hours post-noise exposure to observe if there was expression of p53 protein in injured hair cells. Results Three hours after noise exposure, the outer hair cells at the end of basal turn and beginning of second turn were destroyed first with a character of nuclear condensation. Six hours post-noise exposure, many hair cells in the center of damage region had nuclear fragmentations, and the damaging area expanded towards to basal turn and apical turn. Twelve hours after noise exposure, the nucleus in most outer hair cells and inner hair cells at the region of damage center were missing. The nuclear condensation and fragmentation were appeared in hair cells in both sides of the center region of degeneration. P53 immunoreactive products were also found in damaged hair cells, not only in the central damage area, but also in the basal turn and the third turn.</p><p><b>CONCLUSIONS</b>Intensive impulse noise resulted in apoptosis of cochlear hair cells that initiated between the end of basal turn and the beginning of second turn. Hair cell degeneration spread to basal and third turn along the basilar membrane. P53 may play an important role in impulse noise induced-hair cell apoptosis.</p>

Effects of Erlong Zuoci pill and its disassembled prescriptions on gentamicin-induced ototoxicity model in vitro / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro.</p><p><b>METHODS</b>After the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software.</p><p><b>RESULTS</b>GM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05).</p><p><b>CONCLUSIONS</b>ELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.</p>

Ototoxic effects of streptomycin in vestibular organotypic cultures / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the ototoxic effects of streptomycin in vestibular organotypic cultures.</p><p><b>METHODS</b>F344 rats with age at postnatal day three or four were used for this study. The maculae of saccule and utricle were routinely dissected out and cultured with serum-free medium containing various dose of streptomycin for 24 hours. The ciliary turf of vestibular hair cells was stained with fluorescent phalloidin, and its nucleus was stained with to pro-3 DNA probe. The vestibular hair cells were quantitatively counted and photographed under confocal fluorescent microscope.</p><p><b>RESULTS</b>Morphological feature of vestibular hair cells were good in normal control cultures. However, the density of hair cells was decreased in evidence with increase of streptomycin sulfate concentrations. Twenty-four hours after streptomycin cultures, 0.25 mmol/L streptomycin caused a 10% hair cell missing, 50% hair cell loss from 1 mmol/L streptomycin treatment, and more than 75% hair cells gone post-3 mmol/L streptomycin cultures. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in vestibular hair cells, whereas the vestibular supporting cells were normal.</p><p><b>CONCLUSION</b>Streptomycin induced-vestibular hair cells lesion was in a dose dependent manner with nuclear shrinkage and fragmentation. This may suggest that streptomycin leads vestibular hair cell die through apoptosis.</p>

Correlation between reduction of distortion product otoacoustic emission and percentage of outer hair cell missing in chinchillas / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the quantitative relationship between the reduction of distortion product otoacoustic emission (DPOAE) and the percentage of outer hair cell loss.</p><p><b>METHODS</b>Coadministration of cisplatin (0.2 mg/kg) and ethacrynic acid (40 mg/kg) were used to establish a cochlear lesion model in chinchillas. DPOAE was measured before and 1 week, 2 weeks, and 3 weeks later respectively after cisplatin and ethacrynic acid treatment. Animals were terminated 3 weeks after the treatment. Cochlear surface preparations were performed, and the cochlear hair cells were counted through entire length of the cochlea. The correlation between DPOAE reduction and outer hair cell missing was analyzed using Pearson correlation analysis.</p><p><b>RESULTS</b>Cisplatin and ethacrynic acid treatment induced cochlear hair cell lesion that the outer hair cell loss in the cochlea developed in a stereotypic pattern; damage began in the base of the cochlea and progressed towards the apex. Reduction of DPOAE was relatively consistent with outer hair cells loss. On the average, 1% outer hair cells loss may result in 0.24 dB reduction in DPOAE levels. Pearson analysis showed a positive correlation between the reduction in DPOAE and missing of outer hair cells (r = 0.796, P < 0.05).</p><p><b>CONCLUSIONS</b>It may be helpful to evaluate missing percentage of outer hair cells from reduction in DPOAE levels.</p>

Streptomycin-induced apoptosis of rat cochlear hair cell cultured in vitro / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells.</p><p><b>METHODS</b>F344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope.</p><p><b>RESULTS</b>Streptomycin with 1 mmol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated.</p><p><b>CONCLUSIONS</b>Apoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.</p>

PREPARATION OF ~(35)S-LABEJJED CPS_1 cDNA PROBE FOR IN SITU HYBRIDIZATION / 解剖学报

The carbamyl phosphate synthetase I (CPS,) cDNA probe labelled with alpha ~(35)S-dATP was obtained by using modified nick translation technigue. The specific radioactivity of the obtained probe reached 0.9-1.5?10~8cpm/ug DNA. This probe could be satisfactorily used in RNA-DNA in situ hybrioization for detecting the gene expression of CPS_1 in liver tissue sections. The advantages of using ~(35)S instead of ~(32)P and ~3H for labelling DNA probe were discussed.