AIM:To investigate the effect of rhein on bleomycin-induced pulmonary fibrosis and the expression of microRNA-21 (miR-21) and transforming growth factor-β1 (TGF-β1)/Smad signaling molecules in rats.METHODS:A single dose of bleomycin was intratracheal injected into the SD rats to induce pulmonary fibrosis .After injection of bleo-mycin, the rats were randomly divided into low-, medium-and high-dose rhein treatment groups and model group .The rats that were instilled with normal saline intratracheally served as control group .After the treatment for 28 d, the pulmonary pathologic changes were observed under microscope with hematoxylin-eosin staining .The lung coefficient and hydroxypro-line content were also measured .The expression of miR-21 and the mRNA levels of TGF-β1 and Smad7 in the lung tissues were detected by real-time PCR.The protein levels of TGF-β1 and Smad7 were determined by Western blot .RESULTS:Rhein significantly attenuated the experimental alveolitis , pulmonary fibrosis , lung coefficient and hydroxyproline contents in the rats.Rhein obviously decreased the expression of miR-21,and the mRNA and protein levels of TGF-β1, but signifi-cantly increased the mRNA and protein levels of Smad 7 in the lung tissues .CONCLUSION: Rhein effectively prevents bleomycin-induced pulmonary fibrosis by inhibiting the expression of miR-21 and promoting the expression of Smad 7, thus regulating the TGF/Smad signaling pathway to decrease extracellular matrix deposition .

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.
Cricetinae ; Animals ; Humans ; Cricetulus ; CHO Cells ; Epigenesis, Genetic/genetics* ; DNA Methylation ; Gene Expression Regulation ; Recombinant Proteins/genetics*

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.
Adenine/metabolism* ; Adenine Nucleotides ; Adenine Phosphoribosyltransferase/genetics* ; Adenosine Monophosphate ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Recombinant Proteins/genetics*