[Protective effect of iridoid glycosides of radix scrophulariae on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model].

OBJECTIVE: To investigate the regulatory effect of iridoid glycoside of radix scrophulariae (IGRS) on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model. METHODS: Rat pheochromocytoma PC12 cells were pretreated with IGRS (50, 100, 200 µg/mL) for 24h, and the in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was applied. The cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The apoptotic rate was detected by flow cytometry. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C/EBP homologous protein (CHOP), caspase-12 protein, and glucose-regulated protein-78(GRP78)were detected by Western blotting. The mRNA expression levels of sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), 1, 4, 5-triphosphate inositol receptor 1 (IP3R1), and ryanodine receptor 2 (RyR2)were detected by real-time RT-PCR. Free Ca2+ concentration [Ca2+]i was determined by using laser scanning confocal microscopy. RESULTS: The damage caused by OGD/R to PC12 cells was significantly reduced by IGRS, with significant effect on increasing survival rate and reducing LDH release (all P<0.01). The expression of GRP78, CHOP, Bax, and caspase-12 were down-regulated (all P<0.01), and the expression of Bcl-2 and Bcl-2/Bax ratio was up-regulated (all P<0.01); IGRS increased the expression of SERCA2 mRNA in PC12 cells after OGD/R injury (P<0.01), decreased [Ca2+]i and down-regulated the expression of RyR2 mRNA and IP3R1 mRNA. CONCLUSIONS: IGRS has neuroprotective effect, which may alleviate cerebral ischemia-reperfusion injury by regulating SERCA2, maintaining calcium balance, and inhibiting endoplasmic reticulum stress-mediated apoptosis.

[Microscopic and molecular identification of pine needles].

OBJECTIVE: To identify pine needles from different plant origins by microscopic and molecular approaches. METHODS: The characteristics of pine needles of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were investigated via plant morphology and microscopic characteristics. ITS2 and rbcL were analyzed with PCR amplification and bi-directional sequencing. MEGA 6.0 was used to calculate the intra-and inter-specific Kimura-2-Parameter (K2P) distances, and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. RESULTS: There were significant differences in the number and length of pine needles, number of vascular bundles, distribution of stomatal lines, number and distribution of resin channels among three kinds of pine needles. The lengths of ITS2 sequences of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 470, 469 and 470 bp, respectively. The lengths of rbcL sequences in three kinds of pine needles were 553 bp. The intraspecific variation rates of ITS2 sequences in Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 0%, 0.2%, and 2.8%, respectively; and the intraspecific variation rates of rbcL sequences were 0%, 2.4%, and 1.1%, respectively. There was no significant barcoding gap in intraspecific and interspecific genetic distances of ITS2 sequences. The intraspecific and interspecific distances of rbcL sequences were clearly separated in the barcoding gap test. The NJ tree based on rbcL showed that the three pine needles clustered into three separate groups, indicating that rbcL DNA marker could distinguish the Pinus massoniana Lamb., Pinus thunbergii Parl., Pinus armandii Franch. and its close relative species. CONCLUSIONS: s The three types of pine needles can be distinguished accurately and rapidly by microscopic and molecular identification. The study provides methodology and experimental basis for the quality evaluation and classification of pine needles.

Iridoid glycosides from Radix Scrophulariae attenuates focal cerebral ischemia­reperfusion injury via inhibiting endoplasmic reticulum stress­mediated neuronal apoptosis in rats.

Iridoid glycosides of Radix Scrophulariae (IGRS) are a group of the major bioactive components from Radix Scrophulariae with extensive pharmacological activities. The present study investigated the effects of IGRS on cerebral ischemia­reperfusion injury (CIRI) and explored its potential mechanisms of action. A CIRI model in rats was established by occlusion of the right middle cerebral artery for 90 min, followed by 24 h of reperfusion. Prior to surgery, 30, 60 or 120 mg/kg IGRS was administered to the rats once a day for 7 days. Then, the neurological scores, brain edema and volume of the cerebral infarction were measured. The apoptosis index was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The effects of IGRS on the histopathology of the cortex in brain tissues and the endoplasmic reticulum ultrastructure in the hippocampus were analyzed. Finally, the expression of endoplasmic reticulum stress (ERS)­regulating mediators, endoplasmic reticulum chaperone BiP (GRP78), DNA damage­inducible transcript 3 protein (CHOP) and caspase­12, were detected by reverse transcription quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. The volume of cerebral infarction and brain water content in the IGRS­treated groups treated at doses of 60 and 120 mg/kg were decreased significantly compared with the Model group. The neurological scores were also significantly decreased in the IGRS­treated groups. IGRS treatment effectively decreased neuronal apoptosis resulting from CIRI­induced neuron injury. In addition, the histopathological damage and the endoplasmic reticulum ultrastructure injury were partially improved in CIRI rats following IGRS treatment. RT­qPCR and western blot analysis data indicated that IGRS significantly decreased the expression levels of GRP78, CHOP and caspase­12 at both mRNA and protein levels. The results of the present study demonstrated that IGRS exerted a protective effect against CIRI in brain tissue via the inhibition of apoptosis and ERS.