Study on the differential hepatotoxicity of raw polygonum multiflorum and polygonum multiflorum praeparata and its mechanism

Abstract

Background: Polygonum multiflorum (PM), a widely used traditional Chinese medicine herb, is divided into two forms, namely raw polygonum multiflorum (RPM) and polygonum multiflorum praeparata (PMP), according to the processing procedure. Emerging data has revealed the differential hepatotoxicity of RPM and PMP, however, its potential mechanism is still unclear.

Methods: In our study, we investigated the differential hepatotoxicity of RPM and PMP exerted in C57BL/6 mice. First, sera were collected for biochemical analysis and HE staining was applied to examine the morphological alternation of the liver. Then we treated L02 cells with 5 mg / mL of RPM or PMP. The CCK8 and EdU assays were utilized to observe the viability and proliferation of L02 cells. RNA sequencing was performed to explore the expression profile of L02 cells. Western blotting was performed to detect the expression level of ferroptosis-related protein. Flow cytometry was used to evaluate ROS accumulation.

Results: In our study, a significant elevation in serum ALT, AST and TBIL levels was investigated in the RMP group, while no significant differences were observed in the PMP group, compared to that of the CON group. HE staining showed punctate necrosis, inflammatory cell infiltration and structural destruction can be observed in the RPM group, which can be significantly attenuated after processing. In addition, we also found RPM could decrease the viability and proliferation capacity of L02 cells, which can be reversed by ferroptosis inhibitor. RNA sequencing data revealed the adverse effect of PM exerted on the liver is closely associated with ferroptosis. Western blotting assay uncovered the protein level of GPX4, HO-1 and FTL was sharply decreased, while the ROS content was dramatically elevated in L02 cells treated with RPM, which can be partially restored after processing.

Conclusions: The hepatotoxicity induced by RPM was significantly lower than the PMP, and its potential mechanism is associated with ferroptosis.

Keywords: Ferroptosis; Hepatotoxicity; Polygonum multiflorum; Polygonum multiflorum praeparata; Raw polygonum multiflorum.

Fig. 1

Increased liver toxicity was observed in the RPM group compared to that of the PMP group. A-C: Serum TBIL, ALT and AST level (n = 6). ^**P<0.01 vs. the CON group; ^##P<0.01, vs. the RPM group. D: HE staining was used to evaluate the morphological alternation of mice treated with RMP or PMP (n = 6)

Fig. 2

Aggravated damage to L02 cells was observed in the RPM group compared to that of the PMP group. A: Cell viability was measured by the CCK8 assay. ^**P<0.01, vs. the CON group; ^##P<0.01, vs. the RPM group. B-C: The EdU assay was utilized to evaluate the proliferation capacity of L02 cells treated with different concentrations of RPM or PMP for 24 h. ^**P<0.01, vs. the CON group; ^##P<0.01, vs. the RPM group

Fig. 3

Differential transcriptional expression profile of L02 cells treated with RPM and PMP (n = 3). A: Volcano plot of the expression profile in the RPM group compared to the CON group; B: Volcano plot of the expression profile in the PMP group compared to the RPM group. C: Overlaps of the DEGs between the RPM and PMP groups. D: Systematic cluster analysis of intersected genes with a relative expression greater than 1. E: GO enrichment analysis; F: KEGG enrichment pathway analysis. G: The alternation trend of ferroptosis-related genes in the RPM group compared to the CON group; H: The alternation trend of ferroptosis-related genes in the PMP group compared to the RPM group; I: The network diagram of the overlapped genes

Fig. 4

The different effects of RPM and PMP exerted on ferroptosis. A: GPX4, HO-1, FTL, and GSS protein levels in L02 cells treated with RPM or PMP (n = 3); B: ROS accumulation in L02 cells treated with RMP or PMP (n = 3). ^**P<0.01, vs. CON group; ^##P < 0.01, vs. the RPM group

Fig. 5

Ferroptosis inhibitor reduces the hepatotoxicity induced by RPM. A: CCK8 was applied to evaluate the viability of L02 cells treated with RPM plus Fer-1 (n = 6). ^**P<0.01, vs. the CON group; ^##P < 0.01,vs. the RPM group; ^&&P<0.01, vs. the RPM group. B-C: EdU assay was applied to evaluate the proliferation capacity of L02 cells treated with RPM plus Fer-1 (n = 3). ^**P<0.01, vs. the CON group; ^##P < 0.01, vs. the RPM group;^&&P<0.01, vs. the RPM group

Fig. 6

Fer-1 reduces RPM-induced hepatotoxicity by regulating GPX4 expression and ROS accumulation. A: GPX4 protein level in L02 cells treated with Fer-1 and RPM (n = 3). ^**P<0.01, vs. the CON group; ^##P < 0.01, vs. the RPM group; ^&&P<0.01, vs. the RPM group. B: ROS accumulation in L02 cells treated with RPM and Fer-1 (n = 3). ^**P<0.01, vs. the CON group; ^##P < 0.01, vs. the RPM group; ^&&P<0.01, vs. the RPM group

Fig. 7

The potential mechanism of differential hepatotoxicity RPM and PMP exerted on liver damage. PM, especially RPM, could suppress the expression of GPX4, HO-1 and FTL, resulting in ROS accumulation and leading to hepatocyte ferroptosis