Effect of matrine in MAC-T cells and their transcriptome analysis: A basic study

Abstract

Matrine, an alkaloid derived from herbal medicine, has a wide range of biological activities, including antibacterial. Matrine was toxic to multiple cells at high concentrations. Bovine mammary epithelial cells (MAC-T) could be used as model cells for cow breast. Matrine was a feasible option to replace antibiotics in the prevention or treatment of mastitis against the background of prohibiting antibiotics, but the safe concentration of matrine on MAC-T cells and the mechanism of action for matrine at different concentrations were still unclear. In this study, different concentrations of matrine (0.5, 1, 1.5, 2, 2.5 and 3 mg/mL) were used to treat MAC-T cells for various time periods (4, 8, 12, 16 and 24 h) and measure their lactic dehydrogenase (LDH). And then the optimal doses (2 mg/mL) were chosen to detect the apoptosis at various time periods by flow cytometry and transcriptome analysis was performed between the control and 2 mg/mL matrine-treated MAC-T cells for 8 hours. The results showed that matrine was not cytotoxic at 0.5 mg/mL, but it was cytotoxic at 1~3 mg/mL. In addition, matrine induced apoptosis in MAC-T cells at 2 mg/mL and the proportion of apoptosis cells increases with time by flow cytometry. RNA-seq analysis identified 1645 DEGs, 676 of which were expressed up-regulated and 969 were expressed down-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to matrine-induced toxicity and apoptosis, including cytokine-cytokine receptor interaction pathway, viral protein interaction with cytokine and cytokine receptor, P53 and PPAR pathway. We found 7 DEGs associated with matrine toxicity and apoptosis. This study would provide a basis for the safety of matrine in the prevention or treatment of mastitis.

Fig 1. Determination of LDH release amount and apoptosis rate after treatment of MAC-T cells with matrine.

A. MAC-T cells were treated with matrine at a series of concentrations (0.5-3mg/mL) for 4~24 h. The cell cytotoxicity was evaluated by the LDH assay. B. MAC-T cells were treated with matrine at a concentration of 2 mg/mL for 4~24h. Apoptosis detection with Annexin V-FITC/PI double staining in different groups by flow cytometry. The Q1 represents necrotic or mechanical damaged cells. The Q2 region represents late apoptotic cells. The Q3 region represents viable cells, while the Q4 region represents early apoptotic cells. C. Column bar graph of mean cell florescence for late apoptotic cells. The data are presented as the mean ± S.D. of three independent experiments (*p < 0.05).

Fig 2. Transcriptome data analysis.

A.Volcano plot of total differentially expressed genes in matrine-treated MAC-T cells. Log2|Fold Change|>1 and p< = 0.05 are thresholds. Red plots represent up-regulated DEGs and green plots represent down-regulated DEGs. B. GO functional enrichment pathway of matrine-treated groups. C. KEGG enrichment analysis of selected DEGs in matrine-treated groups. D. The protein−protein interaction (PPI) networks of selected DEGs.

Fig 3. Clustering diagram of important differentially expressed genes.

Red represents highly expressed genes and blue represents lowly expressed genes.

Fig 4. The DEGs were verified by qPCR and the effect of matrine on the expressions of apoptosis-related proteins was measured by western blotting.

A. Comparison of the relative expression of DEGs screened by RT-qPCR and RNA-seq for the matrine-treated group. B. matrine-treated group immunoblots showing expression levels of BAX, IL6, TNF, Caspase3, P65, Smad4, and PrP. C. Gray-scale analysis of matrine-treated group showing expression levels of BAX, IL6, TNF, Caspase3, P65, Smad4, and PrP.