MicroRNA-21/PTEN/PI3K/AKT/mTOR Signaling Pathway Contributes To Ursolic Acid(UA) Induced Anti-proliferation Effects In Rat Primary VSMCs Cells

Objective To investigate the quality standard of Arctii Radix. Methods We make use the standard methods in the appendices of 2010 th Chinese Pharmacopoeia to determinate traditional quality guidelines of crude drugs, such as impurity content, water content,total ash content, heavy metal(Pb) and water-soluble extraction. TLC was used to identify qualitatively oleanolic acid that is an ingredient of Arctii Radix and HPLC was utilized to determine the chlorogenic acid of the Arctii Radix, in addition, study on the method of precision, stability, reproducibility, linear regression and recovery rate were assessed. Further more, The distribution status, botanical characters of typical Arctii Radix. were investigated. Results we suggest that the contents of impurity shouldn’t be measured, the water contents shouldn’t be above 20.0%, the contents of total ash shouldn’t be above 8.0%, the contents of heavy metal(Pb) are below 5ppm, the contents of water-soluble extraction should be above 50%. Arctii Radix identification key was established, the fluorescent spots with the same color can be identified on the relevant position of reference substance and control medicinal material. Methodological investigation show that the instrument precision is fine, the sample detection in 10 h was relatively stable, the reproducibility is good, the Chlorogenic acid had a good linear relationship at the range of 0.1408 μg~1.056 μg(y=3156924.9x-26397.4 R2=0.998) and its mean recovery is 99.4%(RSD=1.34%). Conclusion The methods has been proved to be special and accurate, and can be used for the qualitative and quantitative detective the quality control of Arctii Radix,the quality control method was established for the fist time, has been indexed in Chinese herbal medicines standard of Shandong province(2011) and lay the foundation for government standard.Objective To establish primary culture of vascular smooth muscle cell(VSMCs) and 10% fetal calf serum(FBS)induced cellular proliferation model, then study the anti-proliferation effects of ursolic acid(UA). With liposome-mediated transient PTEN overexpression method, study the role of mi R-21/PTEN/PI3K/Akt/m TOR signaling pathway in the anti-proliferation effects of UA in primary VSMCs. Methods Rat primary VSMCs culture was established with tissue adherent method. Cell identity was confirmed with immunofluorescence staining for α- actin. For the FBS induced proliferation model, VSMCs at generation 3-8 were subjected to 10%FBS for 24 h. LDH assay was used to assess potential cytotoxicity by UA treatment; CCK-8 assay was utilized to determine cell proliferation statuses of VSMCs. For proliferation assay, VSMCs were treated as follows: control,model, mi RNA-21 inhibitor and corresponding scramble control for 60 h, or UA(10, 20 and 30 umol/L) for 48 h.Total incubation time was identical. Realtime RT-PCR was used to assess the expression level of mi RNA-21 and PTEN, western blotting was utilized to investigate the protein level of PTEN. Furthermore, PTEN was overexpressed in VSMCs with liposome transient transfection for 6h. Western blotting was used to assess the expression levels of PTEN and PI3 K. To assess the role of PI3K/Akt signaling pathway in UA mediated anti-proliferation effect, VSMCs were pretreated with UA(10, 20 and 30 umol/L) for 12 h, or with PI3 K inhibitor LY290042 for 30 min, or with Akt inhibitor MK2206 for 24 h, then incubated with 10% FBS for 24 h. The initial and termination time points were identical across groups. Real-time RT-PCR was used to measure mi RNA-21 and PTEN m RNA levels; western blotting was used for assessment of PTEN, PI3 K, Akt and m TOR protein expression levels. Results The primary culture identity was confirmed and purity was estimated to be greater than 98% as indicated by immunofluorescence staining results. 10% FBS treatment effectively induced cell proliferation to429% as compared to control cells. LDH assay demonstrated that UA does not exhibit cytotoxicity to VSMCs at doses equal or lower than 30 umol/L. Cell proliferation data measured with CCK-8 indicated significant anti-proliferation effects of UA as well as LY290042 and MK2206. 10, 20 and 30 umol/L UA inhibited VSMCs proliferation dose-dependently. mi RNA-21 inhibitor treatment decreased the expression level of mi RNA-21,while increased the expression level of PTEN. Overexpression of PTEN resulted in decrease of the expression level of PI3 K. UA treatment effectively increased the expression of PTEN relative to model group, while decreased the expression of PI3K/Akt/m TOR. PI3 K inhibitor LY290042 decreased the expression level of PI3K/Akt/m TOR, while Akt inhibitor MK2206 decreased the expression levels of Akt/m TOR but not PI3 K.Conclusions We successfully reproduced the 10% FBS induced rat primary VSMCs proliferation model. UA does not exhibit cytotoxicity at doses equal or lower than 30 umol/L. 10, 20 or 30 umol/L UA exhibit anti-proliferation effects in VSMCs via mi RNA-21/PTEN/PI3K/Akt/m TOR does-dependently. mi RNA-21 downregulate PTEN,while PTEN in turn downregulate PI3K/Akt/m TOR.