Antioxidant mechanism of gastrodin combined with isorhynchophylline in inhibiting MPP~+-induced apoptosis of PC12 cells / 中国中药杂志

Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis is the most frequently used herbal pair in the treatment of Parkinson's disease(PD). Gastrodin and isorhynchophylline are important components of Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis herb pair with anti-Parkinson mechanism. This study aimed to investigate the effect of gastrodin combined with isorhynchophylline on 1-methyl-4-phenylpyridinium(MPP~+)-induced apoptosis of PC12 cells and their antioxidant mechanism. The leakage of lactate dehydrogenase(LDH) from cells to media was analyzed by spectrophotometry. Apoptotic cells were labeled with Annexin V-fluorescein isothiocyanate(FITC) and propidium iodide(PI) and analyzed by flow cytometry. The cell cycle was analyzed using propidium iodide(PI) staining. Lipid peroxidation(LPO) level was analyzed by spectrophotometry. The mRNA expression of caspase-3 was examined by Real-time RT-PCR. The protein expressions of heme oxygenase 1(HO-1) and NADPH: quinoneoxidore-ductase 1(NQO-1) were determined by Western blot. Gastrodin combined with isorhynchophylline reduced the percentage of Annexin V-positive cells and cell cycle arrest in MPP~+-induced PC12 cells. Gastrodin combined with isorhynchophylline down-regulated the mRNA expression of caspase-3, up-regulated the protein expressions of HO-1 and NQO-1, and reduced LPO content in MPP~+-induced PC12 cells. PD98059, LY294002 or LiCl could partially reverse these changes pretreated with gastrodin combined with isorhynchophylline, suggesting that gastrodin combined with isorhynchophylline inhibited MPP~+-induced apoptosis of PC12 cells and oxidative stress through ERK1/2 and PI3 K/GSK-3β signal pathways. Our experiments showed that gastrodin combined with isorhynchophylline could down-re-gulate the mRNA expression of caspase-3 and up-regulate the protein expressions of HO-1 and NQO-1, so as to reduce oxidative stress and inhibit apoptosis.

Differential mechanism of toxicity in normal and blood-stasis mice following Rhizoma Curcumae exposure based on "YOU-GU-WU-YUN" theory / 药学学报

We were interested in ascertaining differences in developmental neurotoxicity in normal and blood-stasis pregnant mice administered orally Rhizoma Curcumae and the underlying molecular biology mechanisms of any differences. To answer these questions, a blood stasis model was induced by being immersion in ice water. C57BL/6 mice with blood stasis, normal C57BL/6 mice, Nrf2 knock out (KO) mice with blood stasis were randomized into control groups and Rhizoma Curcumae exposure groups. The pregnant mice were administered Rhizoma Curcumae during pregnant day 5 to day 18. The neurodevelopment reflex was examined by the positive occurring time of avoidance precipice reflex tests. Measurement of glutathione (GSH) in brain of the offspring was performed by colorimetric assays. Transcription factor NF-E2-related factor 2 (Nrf2), glutamate cysteine ligase catalytic subunit (GCLc), and glutamate cysteine ligase modifier subunit (GCLm) mRNA and protein expression in brain of the offspring were examined by real-time RT-PCR and Western blot, respectively. All animal care and experiments procedures were reviewed and approved by the Animal Care and Use Committee of Qiqihar Medical College. Our results demonstrated for the first time evidence that C57BL/6 mice treated with Rhizoma Curcumae (10.0 g·kg-1) extended the positive occurring time of avoidance precipice reflex tests of offspring mice compared with the normal control group (P<0.05). We could not find any significant change in that of blood-stasis pregnant mice offspring compared with the normal control group (P>0.05). Compared with the normal control group, level of glutathione, mRNA and protein expression of Nrf2, GCLc, and GCLm significantly increased in brain of the offspring of blood-stasis pregnant mice (all P<0.05). However, mice treated with Rhizoma Curcumae (10.0 g·kg-1) did not change those of offspring (all P>0.05). Knock out Nrf2 using CRISPR/Cas9 extended the positive occurring time of avoidance precipice reflex tests of offspring of blood-stasis pregnant mice (P<0.05). To conclude, developmental neurotoxicity of the blood-stasis pregnant mice to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Cold-induced Nrf2 activation has important roles in "YOU-GU-WU-YUN" phenomenon of Rhizoma Curcumae.

Effect of maternal exposure to Curcumae Rhizoma during pregnancy on neurodevelopment and apoptosis mechanism in offspring / 中国中药杂志

Curcumae Rhizoma is a Chinese medicinal herb that is contraindicated during pregnancy. Cold-congelation and blood-stasis are corresponding syndromes to Curcumae Rhizoma. Whether syndrome-based treatment is associated with developmental neurotoxicity of Curcumae Rhizoma remains to be unclear. To verify the theory of traditional Chinese medicine of "syndrome-based treatment during pregnancy", the present study induced the mice blood stasis model by immersing mice in ice water. Pregnant C57 BL/6 wild type(WT) mice and pregnant Nrf2 knock out(KO) mice were randomly divided into control groups and Rhizoma Curcumae exposure groups. The mice were exposed to Rhizoma Curcumae during day 5 to day 18 after pregnancy. The neurodevelopment was examined to evaluate the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice exposed to Rhizoma Curcumae. caspase-3 and caspase-9 activity in brain of the offspring were measured by colorimetric assays. Bcl-2 mRNA and protein expression in brain of the offspring were examined by Real-time RT-PCR and Western blot, respectively. According to the findings, C57 BL/6 mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) had a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring, compared with the normal control group, but with no significant change in those of blood-stasis pregnant mice offspring. However, mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) showed no change in Bcl-2 gene expression and p38 MAPK phosphorylation in brain of the offspring. Nrf2 gene knockout using CRISPR/Cas9 resulted in a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring. In conclusion, developmental neurotoxicity of the blood-stasis pregnant mice exposed to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Nrf2 activation involved in the phenomenon of Rhizoma Curcumae of "syndrome-based treatment during pregnancy", but the upstream signal pathway mechanism value shall be further investigated.