ABSTRACT
Objective::To investigate the role and mechanism of Testudinis Carapax et Plastrum aqueous extract in promoting osteogenic differentiation of mouse preosteoblast cell line(MC3T3-E1) by regulating nuclear transcription factor-κB(NF-κB) inflammation microenvironment. Method::MC3T3-E1 cells were cultured in vitro, and osteogenic induction (OI) was performed. Testudinis Carapax et Plastrum was prepared and treated the cells. Cells were devided into control group, osteogenic induction group and Testudinis Carapax et Plastrum (20 mg·L-1)with osteogenic induction group. The proliferation of MC3T3-E1 was detected by cell counting kit-8 (CCK-8), and the optimum concentration of intervention was determined. MC3T3-E1 differentiation and osteogenic mineralization were assayed using alkaline phosphatase (ALP) and Alizarin red staining (ARS), respectively. The expressions of NF-κB p65, NF-κB p105, interleukin-6(IL-6), ALP and Collagen-Ⅰ(COL-Ⅰ) mRNA were detected by Real-time PCR. Result::The results of CCK-8 showed that the proliferation of MC3T3-E1 did not change statistically with time, but it showed an upward trend, while the proliferation at 20 mg·L-1 was more obvious than other groups. The ALP and ARS showed that the positive staining rate of osteogenic induction group and Testudinis Carapax et Plastrum with osteogenic induction group were higher than control group.Real-time PCR results showed that on the 7th day in culture, the expression of NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01), and the expression of ALP and COL-Ⅰ mRNA was significantly upregulated(P<0.05), on the 14th day, the expression of NF-κB p65, NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01). The expression of ALP and COL-Ⅰ mRNA was significantly increased (P<0.05, P<0.01). Conclusion::Testudinis Carapax et Plastrum aqueous extract can promote osteogenic differentiation of MC3T3-E1 via a mechanism associated with the regulation of inhibition of NF-κB inflammatory microenvironment.
ABSTRACT
OBJECTIVE: To explore the characteristic of Chinese herbal medicine Testudinis carapax et Plastrum by the multiple PCR technique. METHODS: The salting out method was modified to extract mitochondrial DNA (mtDNA) from Testudinis carapax et Plastrum and their counterfeits. The sequences of cytochrome b (Cyt b) and cytochrome c oxidase subunit I (Col) genes were downloaded from the GenBank database, and two specific pairs of primers were designed with Premier 5.0. The genes of Testudinis carapax et Plastrum were amplified with multiple PCR and sequenced. RESULTS: The size of mtDNA got by salting out method was 16.6×103 bp, and two bright stripes between 300-500 bp were shown in agarose gel electrophoresis of authentic Testudinis carapax et Plastrum, only one or no stripe appeared for the counterfeits. CONCLUSION: The multiple PCR technique is specific, simple, and accurate for differentiation of authentic Testudinis carapax et Plastrum from their counterfeits.
ABSTRACT
OBJECTIVE: To establish a practical, convenient and accurate method of DNA molecular marker for the identification of Testudinis Carapax et Plastrum. METHODS: Phenol extraction method and salting-out method were used to extract mtDNA from Testudinis Carapax et Plastrum and its adulterants. Cyt b mtDNA sequences of Chinemys reevesii and other turtle species were downloaded from GenBank. Species specific PCR primers were designed according to the differential DNA fragments of Cyt b genes, and Cyt b gene segment was amplified by PCR technique and sequenced. RESULTS: The complete 16.6 kb mtDNA was extracted from all samples by the two extraction methods. The specific primers could only amplify the Cyt b gene sequence of Chinemys reevesii, and its fragment was about 319 bp. The result of sequencing indicated that the homologous similarity was 100% with Chinemys reevesii. CONCLUSION: Salting-out method is suitable for the DNA extraction from Testudinis Carapax et Plastrum. The primers are specific to Chinemys reevesii and can be used to distinguish Testudinis Carapax et Plastrum from its adulterants. Copyright 2012 by the Chinese Pharmaceutical Association.
ABSTRACT
Objective: To observe the protection of Testudinis Carapax et Plastrum extracts (TCPE) on serum starvation-induced PC12 cell apoptosis and explore its mechanism. Methods: The PC12 apoptosis model was established by serum starvation for 3 d. The cells were randomly divided into four groups: control group, model group, low-dose and high-dose (3 and 30 μg/mL) TCPE groups. In the three days of the treatment, cell absorbance was determined by MTT, ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry (FCM), Caspase-3, BMP4, BMPR-IA, and p-Smad1/5/8 signaling molecular expression were detected by Western blotting, and the anti-apoptotic effect of TCPE was observed after blocking BMPs signal pathway. Semi-quantitative analysis of bands was carried out by Bio-Rad Quantity One gel analysis system. Results: MTT and FCM analyses demonstrated that TCPE could increase PC12 cell viability and decrease their apoptotic ratios in a dose dependent manner. Western blotting results showed that TCPE could decrease Caspase-3 expression, promote the expression of BMP4, BMPR-IA, and p-Smad1/5/8. There was statistically significant difference between TCPE (3 and 30 μg/mL) groups and model group (P<0.05, P<0.01) in all above results. While TCPE had no effect on the expression of BMP2, BMP7, and BMPR-II. BMPR-IB hadn't been detected. The anti-apoptotic activity was partially mitigated by neutralizing BMP4 antibody. Conclusion: TCPE has the capacity to inhibit the apoptosis of PC12 induced by serum starvation in a dose dependent manner and its mechanism may be associated with partially activating and up-regulating the expression of BMP4 signaling pathway.