Protective effects of berberine against ß-amyloid-induced neurotoxicity in HT22 cells via the Nrf2/HO-1 pathway.

Neuronal apoptosis has been found to have a pivotal role in the course of Alzheimer's disease (AD). Berberine (BBR), a potent antioxidant, occurs in plants such as Berberis, Phellodendron chinense, and Hydrastis canadensis. In this study, a neuronal apoptotic model was established in vitro using HT22 cells induced by Aß25-35 to explore whether BBR contributes to protecting neurons against Aß25-35-induced neurotoxicity, as well as its potential mechanisms. BBR was applied to HT22 cells for 1 h prior to exposing the cells to Aß25-35 for 24 h. A CCK-8 assay was utilized to assess cell viability, and Annexin V - fluorescein isothiocyanate (FITC)/propidium iodide and Hoechst 33342 fluorescence staining were used to measure the rate of cell apoptosis. Existing scientific literature was also reviewed to further determine the effects of BBR on ROS production and mitochondrial function in HT22 cells. Furthermore, the expressions of proteins, including cytochrome C, cleaved caspase-3, p-p65, p65, and Nrf2/HO-1 antioxidant axis were assessed by Western blotting. The data indicated that BBR markedly improved cell viability, inhibited apoptosis and intracellular ROS levels, improved mitochondrial membrane potentials, decreased the rate of p-p65/p65, cytochrome C, and cleaved caspase-3, and intensified the activity of Nrf2/HO-1 antioxidants in HT22 cells. Overall, the findings indicated that BBR provides a certain level of neuroprotectiveness in HT22 cells exposed to Aß25-35 via relieving oxidative stress, as well as by restraining the mitochondrial pathway of cellular apoptosis. In addition, the restraint of NF-κB activity and sensitization of the Nrf2/HO-1 antioxidant axis, which together are intimately involved in the neuroprotection of BBR, may be possible mechanisms accounting for its effectiveness against Aß25-35in vitro.

[Determination of volatile nitrosamines in urine through gas chromatography-mass spectrometry].

OBJECTIVE: To establish a detection method based on gas chromatography-mass spectrometry (GC-MS) for concentrations of volatile nitrosamine compounds in urine, and apply it to the test of real samples. METHODS: Target compounds dichloromethane in urine samples was extracted with dichloromethane through liquid-liquid extraction, then the dichloromethane extract was filtrated, evaporated with nitrogen at 40°C to dryness, and the volume was set with 0.2 ml dichloromethane. Analysis of nine volatile nitroso-compounds were performed with GC-MS under selected ion monitoring mode, external reference method was used for quantification, and the detection limit, repeatability and sensitivity were evaluated. In addition, nine volatile nitroso-compounds of 92 urine samples in a town of Anhui province were measured. RESULTS: A good linear range of 2 - 200 ng/ml (with correlation coefficient 0.9985 - 0.9999) were obtained for the above mentioned nine kinds of analyte, and the lowest examination concentration was 0.05 - 0.50 ng/ml. The addition standard recoveries were 68%-102% with the RSD of 0.4% - 5.5% (n = 3). The detection limits were 0.001 - 0.013 ng/ml urine. The detection rate of N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosopiperidine (NPIP), N-nitrosodi-n-butylamine (NDBA) and N-nitrosodiphenylamine (NDPhA) were 71% (65), 74% (68), 65% (60), 80% (73), 92% (85), 78% (72), 76% (70), 87% (80), 98% (90), respectively, with the results (0.27 ± 0.12), (0.75 ± 0.29), (0.06 ± 0.02), (0.16 ± 0.07), (23.66 ± 5.18), (1.01 ± 0.35), (0.38 ± 0.11), (2.47 ± 0.52) and (15.13 ± 3.48) nmol/g creatinine. CONCLUSIONS: A gas chromatography-mass spectrometry detect method was developed for low level volatile nitrosamines in urine samples.

Geniposide attenuates Aß25-35-induced neurotoxicity via the TLR4/NF-κB pathway in HT22 cells.

Alzheimer's disease (AD), a neurodegenerative disorder, is marked by the accumulation of amyloid-ß (Aß) and neuroinflammation which promote the development of AD. Geniposide, the main ingredient isolated from Chinese herbal medicine Gardenia jasminoides Ellis, has a variety of pharmacological functions such as anti-apoptosis and anti-inflammatory activity. Hence, we estimated the inflammatory cytotoxicity caused by Aß25-35 and the neuroprotective effects of geniposide in HT22 cells. In this research, following incubation with Aß25-35 (40 µM, 24 h) in HT22 cells, the methylthiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) release assays showed that the cell survival rate was significantly decreased. In contrast, the reactive oxygen species (ROS) assay indicated that Aß25-35 enhanced ROS accumulation and apoptosis showed in both hoechst 33342 staining and annexin V-FITC/PI double staining. And then, immunofluorescence test revealed that Aß25-35 promoted p65 to transfer into the nucleus indicating p65 was activated by Aß25-35. Moreover, western blot analysis proved that Aß25-35 increased the expression of nitric oxide species (iNOS), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-1ß (IL-1ß). Simultaneously, Aß25-35 also promoted the expression of toll-like receptor 4 (TLR4), p-p65 and p-IκB-α accompanied with the increase in the level of beta-secretase 1 (BACE1) and caspase-3 which further supported Aß25-35 induced apoptosis and inflammation. Fortunately, this up-regulation was reversed by geniposide. In conclusion, our data suggest that geniposide can alleviate Aß25-35-induced inflammatory response to protect neurons, which is possibly involved with the inhibition of the TLR4/NF-κB pathway in HT22 cells. Geniposide may be the latent treatment for AD induced by neuroinflammation and apoptosis.

[Effects of Naohuandan Recipe on learning and memory abilities of SAM-P/8 mice and its role in anti-oxidation and anti-apoptosis].

OBJECTIVE: To investigate the effects of Naohuandan Recipe on learning and memory abilities of SAM-P/8 mice and its role in anti-oxidation and anti-apoptosis. METHODS: Forty SAM-P/8 mice were randomly divided into four groups, which were untreated (normal saline-treated) group, Yinkeluo Tablets (extracts of gingko leaf)-treated group, low-dose Naohuandan Recipe-treated group and high-dose Naohuandan Recipe-treated group. Mice in these groups were given corresponding drugs orally for 105 days. Then the performances of learning and memory of mice were tested by a step-down passive avoidance task and a Y-maze test. The serum levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were detected. The expression level of bcl-xl mRNA in cerebral cortex and hippocampus of mice was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The performances of learning and memory in the Yinkeluo Tablets-treated group, low- and high-dose Naohuandan Recipe-treated groups were significantly improved as compared with those in the untreated group (P<0.05 or P<0.01), and such performance was the best in the high-dose Naohuandan Recipe-treated group among these four groups (P<0.01). The serum levels of SOD and GSH-Px and the expression of bcl-xl mRNA in cerebral cortex and hippocampus of mice in the Yinkeluo Tablets-treated group, low- and high-dose Naohuandan Recipe-treated groups were also significantly higher than those in the untreated group (P<0.05 or P<0.01), while the serum level of MDA in the untreated group was higher than that in the other three groups (P<0.01). CONCLUSION: Naohuandan Recipe can improve learning and memory abilities of SAM-P/8 mice, and this effect may be related to its anti-oxidation efficacy and enhancement of expression level of bcl-xl mRNA.

Clinical observation on the acupuncture treatment in patients with urinary retention after radical hysterectomy.

OBJECTIVE: To observe the acupuncture therapy effect on the urinary retention after radical: hysterectomy. METHODS METHODS: Eighty cases of urinary retention after radical hysterectomy were randomly assigned to: the treatment group and control group according to the random number table method, 40 cases in each group. From the 15th day post operation, the patients in the two groups started to be treated and 7 days as a course with 5 days treatment and 2 days interval. The treatment group applied acupuncture with modalities of common needling on Zusanli (ST36) bilaterally, electroacupuncture on Sanyinjiao (SP6), Shuidao (ST28), and Scalp Reproduction Area bilaterally, moxibustion on Shenque (CV8). The control group applied acupoint injection with vitamin B B12 12, and Sanyinjiao and Zusanli were selected. Take turns on both sides. The courses for the recovery, of bladder function and residual urine volume for those who had voluntary micturition more than 200 mL after the first and second course of treatment were compared between the two groups. RESULTS: Within 1 course and 2: courses of treatment, the patients with bladder function recovery in the treatment group were 21 (21/40) and 36 (36/40), and those in the control group were 12 (12/40) and 29 (29/40), both with a significant difference (P<0.05). After the first course and second course, residual urine volume for those who had voluntary micturition more than 200 mL in the treatment group was 91.7 ± 17.5 mL and 93.5 ± 15.5 mL, in the control group 102.4 ± 13.7 mL and 102.5 ± 15.7 mL, both with a significant difference (P<0.05). CONCLUSIONS: Combination of acupuncture: modalities was better than acupoint injection for the recovery of bladder function in urinary retention after radical hysterectomy. It would shorten the course of treatment and get a better recovery, decrease urinary retention cases of refractoriness, and extending the treatment could raise the healing rate of urinary retention.