Raw SO (the dried aerial part of Siegesbeckia orientalis L.) was collected from Ganzhou (Jiangxi province, China). Rice wine was purchased from Zhejiang Tapai Shaoxing Wine Co., Ltd. (Zhejiang province, China). LC–MS grade acetonitrile, methanol, and formic acid (FA) were purchased from CNW Technologies (Germany). Kirenol was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Adonitol was purchased from Sigma-Aldrich (MO, United States). Milli-Q water was prepared using a Milli-Q system (Millipore, MA, United States). The commercial kits including creatine kinase (CK), lactate dehydrogenase (LDH), urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Guangdong province, China). Malondialdehyde (MDA) was purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu province, China).

The authentication of SO was confirmed in accordance with the corresponding monograph in CP 2020 (2020 edition of CP) by the corresponding author. Voucher specimens of raw SO (No. 20180510) and rice wine were deposited at the International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine.

PSO preparation: As shown in our previous study, 100 ml of rice wine (containing 15% ethanol): water/20:80 (v/v) was sprayed onto 100 g SO and allowed to be completely absorbed in a covered container for 1.5 h. Subsequently, the mixture was steamed for 5 h, cooled, and then dried at 40°C in a Memmert UFE500 oven (Su et al., 2014).

Raw SO and PSO extracts preparation: Raw SO or PSO (500 g) was reflux-extracted twice with water (1:10, w/v) for 2 h each. The combined extracts were filtered after cooling and then concentrated under reduced pressure to remove the water. The powdered SO (yield: 18.5%) and PSO (yield: 14.9%) extracts were obtained by lyophilizing of the concentrated samples with a VirTis Freeze Dryer (The VirTis Company, New York, United States). Powdered SO or PSO was dissolved in water to prepare raw SO and PSO solution. Detailed information of the high performance liquid chromatography (HPLC) analysis was shown in Supplementary Materials. Results showed that the contents of kirenol in raw SO and PSO were 0.13 and 0.07%, respectively (Supplementary Figure S1).

A total of 24 male SD rats (200 ± 20 g, 6–8 weeks) were obtained from the Laboratory Animal Center of Southern Medical University [License number: SCXK (GZ)2016-0041; Guangzhou, China] and kept in the animal laboratory [License number: SYXK (GZ) 2019-0144] at the International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine (Guangzhou, China). Animal experiments were approved by the Guangzhou University of Chinese Medicine Animal Care and Use Committee (Guangzhou, China) and conducted according to the ethical standards and national guidelines. Every effort was made to reduce the number of animals used and minimize their pain and distress. After one week of acclimatization, rats were randomly divided into three groups (n = 8), and daily intragastrically administered an equal volume of the vehicle, raw SO or PSO extract at a dosage of 5 g/kg/day for 6 months. The experimental design was shown in Supplementary Figure S2.

Blood samples were collected from the retro-orbital venous plexus each month. The blood samples were centrifuged at 3500 rpm for 10 min after standing for 2 h at 4°C. The serum was then transferred into new tubes and stored at −80°C for further analysis. A portion of the collected serum was used for routine biochemical analyses of the creatine kinase (CK), lactate dehydrogenase (LDH), urea, aspartate transaminase (AST), alanine transaminase (ALT), and plasma malondialdehyde (MDA) levels according to the manufacturer’s instructions of the respective commercial assay kits. At the end of the experiment, all rats were sacrificed followed by blood collection. Fresh cardiac, hepatic, lung, and renal tissues were dissected and fixed in 10% neutral buffered formaldehyde at 4°C for paraffin embedment. Organ samples (4 μm) were sectioned and stained with H&E, and finally examined under light microscopy.

100 μL of serum was transferred and 400 μL extract solution (acetonitrile: methanol = 1:1) containing internal standard (adonitol, 1 μg/ml) was added. After 30 s vortex, samples were sonicated for 5 min in an ice-water bath. Then the samples were incubated at −40°C for 1 h and centrifuged at 10,000 rpm for 15 min at 4°C. 400 μL of supernatant was transferred to a fresh tube and dried in a vacuum concentrator at 37°C. Then, the dried samples were reconstituted in 100 μL of 50% acetonitrile by sonication on ice for 10 min. The constitution was then centrifuged at 13000 rpm for 15 min at 4°C, and 60 μL of supernatant was transferred to a fresh glass vial for LC/MS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all the samples.

The UHPLC separation was carried out using an ExionLC Infinity series UHPLC System (AB Sciex), equipped with a UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water A) and acetonitrile B). The analysis was carried with an elution gradient as follows: 0–0.5 min, 95%B; 0.5–7.0 min, 95%–65% B; 7.0–8.0 min, 65%–40% B; 8.0–9.0 min, 40% B; 9.0–9.1 min, 40–95% B; 9.1–12.0 min, 95% B. The column temperature was 25°C. The auto-sampler temperature was 4°C, and the injection volume was 2 μL (pos) or 3 μL (neg), respectively.

The TripleTOF 5600 mass spectrometry (AB Sciex) was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at collision energy (CE) of 30 eV. The cycle time was 0.56 s. ESI source conditions were set as following: gas 1 as 60 psi, gas 2 as 60 psi, curtain gas as 35 psi, source temperature as 600°C, declustering potential as 60 V, and ion spray voltage floating (ISVF) as 5,000 V or −4,000 V in positive or negative modes, respectively.

The pooled quality control (QC) sample was analyzed at the beginning, the end, and randomly through the analytical run to monitor the stability of sequence analysis. The typical batch sequence of serum samples consisting of consecutive analysis cycles of 1 QC serum sample (at the beginning of the study) follow by 8 unknown serum samples. Meanwhile, samples were analyzed in a random order for a normal good practice. An identical sequence was repeated to complete the total set of injections (n = 28, including QCs) analyzed in less than one day per mode.

Rat serum samples were analyzed by an untargeted metabolomics approach based on UPLC-Q-TOF/MS. The MS raw data (.wiff) files were converted to the mzXML format by ProteoWizard, and processed by R package XCMS (version 3.2). Principle component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA) were used to assess the data. Major metabolites were screened and identified using in-house libraries and the METLIN (http://metlin.scripps.edu) database, the MassBank database (http://www.massbank.jp/), the Human Metabolome Database (HMDB) (http://www.hmdb.ca/), or the LipidMaps database (http://www.lipidmaps.org) based on accurate mass measurements (mass errors <5 ppm) and MS/MS fragment information.

Results of the biochemical assays were presented as Mean ± SEM. These data were analyzed by one-way ANOVA followed by the Dunnett’s multiple comparisons using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, United States). p < 0.05 was considered statistically significant.

The serum levels of AST, ALT, Urea, CK, and LDH were measured in each rat every month. ALT and AST are the markers of hepatic damage; Urea is a marker of renal damage; CK is a marker of cardiac damage. However, currently, no specific markers were used for evaluating the lung damage. It is known when an organ is damaged, LDH will be released into the circulation, which is known as a biomarker for tissue damage. Moreover, it has also been reported that the measurement of LDH activity in lung lavage fluid can assess the extent of cell damage (Gao et al., 2013). Here, we found that the serum level of LDH in raw SO group was significantly higher than that in the control group after 6-months treatment (p < 0.01). It is worth noting that, the LDH level in the PSO group was lower than that in the raw SO group (p < 0.05) (Figure 1), suggesting that processing significantly reduced the LDH level in the serum. Although raw SO increased the levels of CK and AST in the serum when compared to the control groups, they did not reach statistical significance. In addition, we also found that both raw SO and PSO did not significantly affect the serum levels of ALT, AST, Urea, and CK in the earlier stage of the treatment, suggesting that the treatments at the early stage have no obvious liver, kidney and heart toxicities were observed in these rats (Supplementary Figure S3).

H&E staining was used to further examine the treatment-induced toxicities in heart, liver, lung, and kidney tissues. We found that histopathological damages were observed in the lung tissues of the raw SO-treated rats as indicated by a lot of inflammatory cells infiltration, and along with the cells edema; while, the damages were relatively mild in PSO-treated rats. Liver and heart sections showed little pathological damages in the raw SO group (Figure 2), suggesting that long-term administration of raw SO may not cause obvious damages in the liver and heart when compared to the lung. Combining the pathological examination and the biochemical results, we strongly suggest that raw SO mainly causes pulmonary toxicity, which can be significantly reduced by processing.

Total ion chromatogram (TIC) chromatograms of the serum samples were obtained from vehicle-, raw SO-, and PSO-treated rats (Supplementary Figure S4). Metabolomics chromatograms obtained through LC-Q-TOF/MS using the ESI⁺ and ESI⁻ modes revealed distinct differences. In total, 32 altered metabolites were authentically identified, including amino acids, lipids, hydrocarbons, and nucleotides. The heatmap figure showed the detailed information (Supplementary Figure S5). Based on the information obtained from LC-Q-TOF/MS, general OPLS-DA and PCA models were calculated to detect the significantly changed metabolites (VIP>1 or p < 0.05) among the control, raw SO and PSO groups. PCA scores (PC1: 14.4%, PC2: 12.4%, PC3: 9.32%) showed that the three groups were clustered (Figure 3A). The multiple pattern recognition method of partial least squares discriminant analysis was adopted based on the basis of the metabolic changes in the three groups as revealed by TIC chromatograms. As shown in the PLS-DA scores plot (Figure 3B, R₂X = 0.41, R₂Y = 0.841, Q₂ = 0.388), we found that the distribution of each group was concentrated, and the distance between the raw SO group and control group was far, while that between the PSO group and the control group was comparatively shorter, suggesting that significant changes of the metabolites in the serum levels are shown after the administration of raw SO in rats.

As shown in Table 1, a total of 32 metabolites were significantly changed when compared to the control group, among them, 10 metabolites were significantly downregulated including LysoPC(20:4), LysoPC(22:6), and glucosylsphingosine, while 22 metabolites were significantly upregulated including L-glutamine, L-aspartic acid, and phosphorylcholine. For the PSO group, we found that 5 metabolites were significantly downregulated including acetylcysteine, D-tryptophan, and gamma-linolenic acid, while 14 metabolites were significantly upregulated including PC (16:0/16:0), PE(16:0/20:1), and PE (22:5/22:6). Interestingly, 14 metabolites which obviously altered in the raw SO group, showed no significant differences in the PSO group, which is similar to the control group. The volcano figure can also show the metabolites fluctuation of various compounds (Figure 4). These altered metabolites may partly explain the toxicity that is caused by raw SO and the toxicity-reducing effect of processing.

To further understand the correlation between the candidate metabolites and the biological association networks, we performed pathway analysis using MetaboAnalyst (https:// www.metaboanalyst.ca/) pathway analysis. As shown in Figure 5, four significant metabolic pathways including arginine biosynthesis, glycerophospholipid metabolism, D-glutamine, and D-glutamate metabolism, and alanine, aspartate, and glutamate metabolism were identified in the serum samples of raw SO-treated rats; while, histidine metabolism, arginine biosynthesis, and D-glutamine and D-glutamate metabolism were identified in the serum samples of PSO-treated rats.

Among them, two characteristic metabolic pathways alanine, aspartate and glutamate metabolism and glycerophospholipid metabolism were only changed in the raw SO group. It was reported that both glycerophospholipid metabolism (Wang et al., 2019) and arginine biosynthesis (Mizrahi and Warner, 2018) are closely related to oxidative stress. Therefore, we speculated that the raw SO–induced oxidative stress response may underlie the pulmonary toxicity. In addition, histidine metabolism was only changed in the PSO group. Studies suggested that histidine, an amino acid that is essential to humans, exerts favorable cytoprotective effects against oxidative stress as have been demonstrated both in vivo and in vitro, which has been shown to scavenging free radicals (Vera-Aviles et al., 2018). Hence, we speculated that free radical scavenging may be responsible for the toxicity-reducing effect of processing.

In an attempt to verify the highlighted biological functions, we examined the MDA contents in the serum samples of vehicle-, raw SO-, and PSO-treated rats. MDA, a classic biomarker of oxidative stress, is produced by free radicals in the body (Lykkesfeldt, 2007). It is also widely used as an indicator of the free radical level. As shown in Figure 6, we found that raw SO treatment significantly increased the serum MDA content when compared to the vehicle control group (p < 0.05), suggesting that raw SO causes oxidative stress in the rats. While the MDA content in the PSO group was lower than that in the raw SO group (p < 0.05), and no significant difference was observed between the control and PSO groups, suggesting that processing reduces the oxidative stress-causing effects of raw SO or processing enabled the herb to possess a free radical scavenging property. These findings also suggest that free radical scavenging may be responsible for the pulmonary toxicity-reducing effect of processing.