Metabolomics reveals the mechanisms for the cardiotoxicity of Pinelliae Rhizoma and the toxicity-reducing effect of processing

Abstract

Pinelliae Rhizoma (PR) is a commonly used Chinese medicinal herb, but it has been frequently reported about its toxicity. According to the traditional Chinese medicine theory, processing can reduce the toxicity of the herbs. Here, we aim to determine if processing reduces the toxicity of raw PR, and to explore the underlying mechanisms of raw PR-induced toxicities and the toxicity-reducing effect of processing. Biochemical and histopathological approaches were used to evaluate the toxicities of raw and processed PR. Rat serum metabolites were analyzed by LC-TOF-MS. Ingenuity pathway analysis of the metabolomics data highlighted the biological pathways and network functions involved in raw PR-induced toxicities and the toxicity-reducing effect of processing, which were verified by molecular approaches. Results showed that raw PR caused cardiotoxicity, and processing reduced the toxicity. Inhibition of mTOR signaling and activation of the TGF-β pathway contributed to raw PR-induced cardiotoxicity, and free radical scavenging might be responsible for the toxicity-reducing effect of processing. Our data shed new light on the mechanisms of raw PR-induced cardiotoxicity and the toxicity-reducing effect of processing. This study provides scientific justifications for the traditional processing theory of PR, and should help in optimizing the processing protocol and clinical combinational application of PR.

Figure 1. Body weights of vehicle-, raw PR- and PRZA-treated rats.

*p < 0.05, **p < 0.01 vs. control; ^##p < 0.01 vs. Raw PR group.

Figure 2. Biochemical parameters in the serum of vehicle-, raw PR- and PRZA-treated rats.

*p < 0.05, **p < 0.01 vs. control; ^#p < 0.05, ^##p < 0.01 vs. Raw PR.

Figure 3. Histopathological examinations of heart, liver, kidney tissues in vehicle-, raw PR- and PRZA-treated rats, H&E staining, 100×.

(A) Heart tissue of control group: normal myocardial fibers in longitudinal section featuring central nuclei and syncytial arrangement of the fibers; (B) Heart tissue of raw PR group: myocardial fibers with loss of cross striations, not clearly visible nuclei, and inflammatory infiltration; (C) Heart tissue of PRZA group: the histopathological changes were milder than raw PR-treated group; (D) Liver tissue of control group; (E) Liver tissue of raw PR group; (F) Liver tissue of PRZA group; (G) Kidney tissue of control group; (H) Kidney tissue of raw PR group; (I) Kidney tissue of PRZA group. (D–I) Liver and kidney sections of both PR and PRZA groups showed no abnormalities. I. Myocardiocyte necrosis; II. Myocardiocyte rupture; III. Inflammatory infiltration.

Figure 4. Results of the multiple pattern recognition of the serum metabolites in control, raw PR and PRZA groups at the time point of day 14

. (red ) Control group, (brown ) Raw PR group, (blue ) PRZA group.

Figure 5. Molecular networks and the canonical pathways in the serum of raw PR- and PRZA-treated rats.

(A) Raw PR group; (B) PRZA group. The network was gained by overlapping raw PR- or PRZA-treated group’s data to control group’s data. Metabolites are represented as nodes, and the biological relationship between two nodes is represented as a line. The colored symbols represent metabolites that occurring in the tested data, while the transparent entries are molecules from the Ingenuity Knowledge Database. Orange symbols represent up-regulated metabolites; green symbols represent down-regulated metabolites. Solid lines between molecules indicate a direct physical relationship between molecules; dotted lines indicate indirect functional relationships.

Figure 6. The protein expression levels in the heart tissues of vehicle-, raw PR- and PRZA-treated rats.

(A) mTOR and phospho-mTOR protein expression levels; (B) TGF-β1 protein expression levels. The protein expression levels (left, representative results) were determined by Western blot analyses and relative band intensities (right) were analyzed by the Image J software. *p < 0.05, vs. control group; ^#p < 0.05 vs. raw PR group.

Figure 7. The MDA contents in the serum of vehicle-, raw PR- and PRZA-treated rats.

**p < 0.01 vs. control.

Figure 8. The characteristic metabolites and the regulated metabolic regulatory networks of raw PR and PRZA.

Red italics molecules represent characteristic metabolites up-regulation in raw PR group and PRZA group; solid lines between molecules indicate a direct transforming relationship, while dotted lines indicate indirect relationships. “f” represents the fold change value of raw PR vs. control or PRZA vs. control.