This study aims to investigate the anticancer effect of Oroxin B (OB) both in vitro and in vivo, and the molecular mechanism involved in microRNA-221 and the PI3K/Akt/PTEN pathway through modulation of apoptosis in Hepatocellular carcinoma (HCC). DEN-induced rats and HepG2 cells based on the microfluidic chip were employed, while the mRNA and protein expression of microRNA-221, PI3K, p-Akt and PTEN were evaluated by RT-PCR and Western blot analysis. Based on Microfluidic Chip and DEN-induced rat model, OB effectively exerts anti-liver cancer effect both in vitro and in vivo, and the expression of miR-221 in OB treated groups was significantly lower than that in the control group (** p < 0.01). The RT-PCR and Western blot results suggested the PI3K mRNA and protein in OB treated groups were both lower than those in control group and indicated the overexpression of PTEN. Therefore, OB effectively exerts anticancer effects by positively regulating the PTEN gene and then inactivating the PI3K/Akt signaling pathway through down-regulating the expression of the microRNA-221, thereby inducing apoptosis of liver cancer cells. This study offers a theoretical evidence for further development and clinical guidance of OB as an anti-tumor agent.

Background: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. Results: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322–163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. Conclusion: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.