Protective effects of Pimpinella diversifolia DC. root essential oil against lipopolysaccharide/D-galactosamine-induced acute liver injury in mice through inhibiting inflammation and reducing oxidative stress

2.1 Reagents

Lipopolysaccharide (LPS) and C7-C40 saturated alkanes standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-acetylcysteine (NAC), Dichloromethane and D-galactosamine (D-GalN) were obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). M−PER mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA). Bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Rockford, USA). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BD mouse inflammation cytometric bead array (CBA) kit (BD Biosciences, San Diego, USA). Antibodies against F4/80 (ab111101), CD11b (ab133357) and Ly6G (ab238132) and HRP-conjugated secondary antibody (ab205719) were purchased from Abcam (Cambridge, MA, USA). Anti-caspase 3 antibody (14220 T) and anti-β-actin antibody (4970S) were obtained from CST (MA, USA). DAB solution was derived from cwbiotech (Beijing, China). TUNEL apoptosis detection kit was purchased from Beyotime biotechnology (Shanghai, China).

2.2 Plant material and preparation of PDREO

The root parts of P. diversifolia were harvested at Shouning County, Ningde City, Fujian Province, China, in September 2021. Samples were dried at 60 °C (Fig. 1A), powdered, and sieved through a 40-mesh screen (Fig. 1B). A voucher specimen (PDR20210909) was stored in the laboratory of the College of Life Science, Zhejiang Chinese Medical University.

Close

Fig. 1

(A) The root part of P. diversifolia. Its powder (B). (C) Its essential oil is extracted by the SDE method.

Export to PPT

PDREO was prepared by a simultaneous distillation–extraction (SDE) method. Briefly, 500 g powder was submitted to SDE in a classical Likens–Nickerson apparatus for 5 h with 5 L water, dichloromethane (100 mL) was used as the extractant. The dichloromethane phase was collected, dried with anhydrous sodium sulfate, and the organic phase was removed under reduced pressure at 25 °C. Finally, 1.41 g of brownish-yellow essential oil was obtained (Fig. 1C).

2.3 Chemical composition

The chemical profile of PDREO was characterized by gas chromatography (GC) as we previously reported (Wang et al., 2022). PDREO was separated using an Agilent Technologies 7890B system (Agilent Technologies, MA, USA) equipped with an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm) coupled to an Agilent 7250 quadrupole time-of-flight mass spectrometry. With helium as carrier gas, the flow rate was 1.0 mL/min. Then, 1 uL sample was injected at a split ratio of 10:1, keep the temperature of injector at 250 °C. The initial column temperature was 50 °C, maintained for 3 min, then increased to 280 °C at a rate of 5 °C/min, and held for 5 min.

The PDREO components were determined by comparing their Kovats retention index (RI) values (calculated using the RI value of a C₇–C₄₀ n-alkane series [O2si, USA] obtained on the HP-5MS column) with the values in the National Institute of Standards and Technology (NIST) database and those reported in the literature. The relative content of each ingredient in PDREO was determined according to the peak area of GC–MS without correction.

2.4 Animals

C57BL/6 mice (male, 18–22 g) were obtained from the Experimental Animal Center of Zhejiang Academy of Medical Sciences (Hangzhou, China). All animals were kept without specific pathogen conditions, with controlled temperature and humidity, 12/12-h light/dark cycle and free access to water and food. The experiment was performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethical Committee of Zhejiang Chinese Medical University (IACUC-20191202–04).

2.5 Establishment of the LPS/D-GalN-induced ALI model

Forty male C57BL/6 mice were randomly divided into five groups: the control group (vehicle), the model group, the N-acetylcysteine (NAC) (300 mg/kg) group, the PDREO (50 mg/kg) group, and the PDREO (150 mg/kg) group. In the NAC and PDREO groups, mice were injected intraperitoneally with NAC or PDREO (both dissolved with PBS containing 1% Tween 80) once a day for three consecutive days, while mice in the control and model groups were injected with vehicle (PBS containing 1% Tween 80). One hour after the last administration, LPS (40 µg/kg, Sigma) and D-GalN (500 mg/kg, Shanghai Macklin Biochemical Co., Ltd.) were injected intraperitoneally to induce ALI. Mice were sacrificed after 6 h. Blood serum was obtained by centrifuging the blood at 1000 g for 15 min after coagulation. Liver tissue was harvested, and liver sample was fixed in a 10% formaldehyde /PBS solution; the remaining parts were snap-frozen in liquid nitrogen, and stored at − 80 °C.

For the animal survival assay, 50 male C57BL/6 mice were randomly divided into five groups, and treated as described above. After LPS (40 µg/kg) and D-GalN (500 mg/kg) were injected, mice were weighed every 24 h and continuously observed for 120 h, and death was recorded.

2.6 Serum biochemistry

Serum AST and ALT activities were determined by the colourimetric method of Reitman and Frankel using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the instructions. According to the method principle described by (POWELL and SMITH, 1954), the ALP level in serum was measured using commercial kit purchased from Nanjing Jiancheng Bioengineering Institute. The levels of serum inflammatory factors, such as TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1), were measured using a BD mouse inflammation cytometric bead array (CBA) kit (BD Biosciences, San Diego, USA) with a slight modification. Briefly, three capture bead populations were mixed together in equal volumes, and 25 μL of each sample was added, following mixed with 25 μL of PE detection reagent. The mixture was incubated for 2 h at room temperature in the dark. The beads were washed with wash buffer, and detected on a Cytoflex S flow cytometer (Beckman, USA). Data was analyzed using FCAP Array software (BD Biosciences).

2.7 Liver index and biochemistry

The liver was weighed and the liver index (liver weight/body weight, mg/kg) was calculated to measure the edema of the liver tissue. According to the method reported by (Maimaitimin et al., 2018), SOD and CAT activities, GSH and MDA contents in the liver were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)r.

2.8 Histological analysis

Fixed liver tissues were embedded in paraffin, cut into 4-µm-thick sections, and stained with hematoxylin and eosin (HE). The severity of liver damage was graded on a four-point scale according to (Lv et al., 2019), where 1-no damage, 2-mild damage, 3-moderate damage, and 4-severe damage.

2.9 Immunochemistry staining

For immunohistochemical studies, sections were subjected to antigen repair in citrate buffer (pH 6.0, 10 mM) and treated in 3% H₂O₂ for 15 min. Then blocked with 2% bovine serum albumin (Amresco, Solon, OH) for 1 h at 25 °C, followed by incubation with primary antibodies against F4/80 (ab111101, Abcam, 1:100), CD11b (ab133357, Abcam, 1:1000), or Ly6G (ab238132, Abcam, 1:1000) overnight at 4 °C. After washing, sections were incubated with HRP-conjugated secondary antibody (ab205719, Abcam, 1:2000) for 1 h at 25 °C. Positive staining was then observed using DAB solution (Cwbiotech, Beijing, China) with hematoxylin counterstaining.

2.10 TUNEL assay

Hepatocyte apoptosis was evaluated by TUNEL assay. A one-step TUNEL apoptosis detection kit (Beyotime biotechnology, China) was used according to the manufacturer's instructions. Positive staining cells were quantified using Image-Pro Plus software (media controlnetics, Rockville, MD) based on three different random fields of each sample.

2.11 Quantitative real-time PCR (qRT-PCR)

Liver tissue (90–100 mg) was homogenized in 1 mL TRIzol reagent (Invitrogen, USA) using a MagNA Lyser (Roche, Germany) at 6000 rpm for 20 s. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions and quantified by spectrophotometry. cDNA was obtained using the High-Capacity cDNA Reverse Transcription Kit (Cwbiotech, Beijing, China). qRT-PCR was performed on an LC480 Real-Time PCR Detection System (Roche, Germany) using SYBR Green PCR Master Mix (Cwbiotech, Beijing, China). Primers were produced by Sangon Biotech (Shanghai, China) and are listed in Table 1. The β-actin gene (B661302, Sangon Biotech, Shanghai) was used as an endogenous reference. Gene expression levels were calculated using the $2^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ method.

2.12 Western blotting analysis

Liver samples were weighted and total proteins were prepared according to the instructions of the M−PER mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, USA). The levels of capspase 3 and cleaved-caspase 3 proteins were measured by immunoblotting with specific antibodies on a simple western system (ProteinSimple, San Jose, CA, USA) according to our previous report (Jiang et al., 2019).

2.13 Statistical analysis

All data are expressed as mean ± SD. Statistical differences among multiple groups were determined by one-way ANOVA followed by Tukey's multiple comparisons test using GraphPad Prism version 8.0 software (San Diego, CA, USA). P < 0.05 were considered to be statistically significant.

3.1 Chemical composition of PDREO

PDREO is a transparent brownish yellow liquid that emits a unique fragrance. It accounts for about 0.28% of the dry weight of the root, which is higher than that of most other Pimpinella species (Tabanca et al., 2005; Tabanca et al., 2004). A total of 27 components (Fig. 2 and Table 2) were identified from PDREO, accounting for 95.74% of the total volatile components, including terpene components accounting for 45.87%, aromatic components accounting for 32.57%, and aliphatic components accounting for 17.30%. The compounds isodaucene, 4-methoxy-2-(3-methyloxiranyl)-phenyl isobutyrate, and 2-methyl-4-methoxy-2-(3-methyl-2-oxiranyl) phenyl butyrate are the main components of PDREO, with contents of 29.97%, 19.53%, and 6.88%, respectively. In addition, the content of the aliphatic compound geijerene and its isomer is also high, accounting for 15.31% of the total essential oil content. Pseudoisoeugenol type phenylpropanoid derivatives are the characteristic components in the essential oils of Pimpinella species. They are mainly distributed in the roots, and the content of these components in the essential oil of some species is more than 80% (Tabanca et al., 2005). The current work also confirmed the presence of pseudoisoeugenol type phenylpropanoid compounds in the root of P. diversifolia, but the number and content of these compounds are significantly lower than those reported by (Bottini et al., 1985). Most of the other components such as geijerene, β-bisabolene, copaene, and β-caryophyllene have been identified and reported in the essential oils of various Pimpinella species (Bottini et al., 1985; Tabanca et al., 2005; Tabanca et al., 2004). However, it is surprising that the compound isocarotene accounts for 29.97% of PDREO, but has not been reported in the essential oils of other Pimpinella species and the essential oil of P. diversifolia leaves from Zhejiang Province (Xu et al., 2012), which may be attributed to the variety of species, different geographical origins, different harvest times, and other factors.

Close

Fig. 2

Total ion current chromatogram of PDREO by GC-Q/TOF-MS.

Export to PPT

3.2 PDREO can effectively reduce the levels of serum biomarkers of liver injury

Serum biochemical indicators, including ALT, AST, and ALP, are important markers to evaluate liver injury (Shi et al., 2010). As depicted in Fig. 3, the serum ALT, AST, and ALP levels in the model group were significantly higher than those in the control group. Treatment with NAC effectively lowered the serum levels of ALT, AST, and ALP. Surprisingly, PDREO (50 mg/kg) significantly reduced the serum levels of ALT, AST, and ALP; those of ALT and ALP were even significantly lower than those of the NAC group. In the PDREO (150 mg/kg) group, the serum levels of ALT and ALP were almost equal to those in the control group. Studies have shown that ALT is mainly located in the liver (Sherman, 1991), while AST is more widely distributed in the liver, heart, bone, kidney, and brain (Cai et al., 2019). Therefore, ALT is a far more specific than AST in diagnosing liver injury (Xu et al., 2015), which indicates that PDREO exhibits potential hepatoprotective activity.

Close

Fig. 3

Effects of PDREO on serum biomarkers of liver injury. (A) ALT. (B) AST. (C) ALP. Data are expressed as mean ± SD (n = 8). ^##P < 0.01 vs. control group; ^**P < 0.01 vs. model group.

Export to PPT

3.3 PDREO can effectively reduce the liver index and ameliorate liver tissue damage

The livers of mice in the control group were pink, with complete structure and no pathological changes (Fig. 4A). In the model group, the liver was dark red and black. Compared with the model group, the liver of the NAC treatment group was significantly lighter but was still dark red. In the PDREO (50 mg/kg) and (150 mg/kg) groups, liver morphology and color were basically similar to those of the control group. HE staining of liver tissue sections more intuitively reflects the pathological changes. As depicted in Fig. 4B, the hepatocytes in the model group were much smaller than in the control group, the space between hepatocytes was enlarged, and massive red blood cells were exuded, which may be one of the reasons for the deepening of liver color. After NAC treatment, the liver tissue lesions were alleviated, but a large number of red blood cells were still visible. The morphology of hepatocytes in the PDREO (50 mg/kg) treatment group was basically normal; only a few red blood cells were occasionally seen. In the PDREO (150 mg/kg) group, no obvious hepatic injury was observed. The HE staining results were basically consistent with the overall color changes in different treatment groups. The liver damage score (Fig. 4C) in the NAC treatment group was improved compared with the model group, but the injury was still significant compared with the control group. However, no statistically significant difference between the PDREO (50 mg/kg) and (150 mg/kg) treatment groups and the control group were observed. The severity of liver tissue lesions and tissue edema is also reflected in the liver index. As shown in Fig. 4D, the liver index was 53.713 ± 3.642 mg/g BW in the model group, which was significantly higher than that of the control group (36.251 ± 1.286 mg/g BW), which was consistent with the above liver color and HE staining results. The liver index was effectively reduced in all treatment groups, and it was significantly lower in the PDREO (150 mg/kg) group than in the NAC treatment group. These data further confirmed the hepatoprotective activity of PDREO.

Close

Fig. 4

Effects of PDREO on (A) liver morphology, (B) liver histopathology, (C) the liver damage score, and (D) the liver index. Data are expressed as mean ± SD (n = 8). ^##P < 0.01 vs. control group; ^**P < 0.01 vs. model group.

Export to PPT

3.4 PDREO can effectively reduce the serum levels of inflammatory cytokines and inhibit the expression of inflammatory genes in liver

Inflammation is a hallmark of ALI (Triantafyllou et al., 2018); therefore, the serum levels of inflammatory cytokines were determined. As shown in Fig. 5A, TNF-α, IL-6, and MCP-1 levels were significantly higher in the model group compared with the control group. NAC treatment effectively reduced the serum levels of all inflammatory cytokines, but these levels were still significantly higher than in the control group. In the PDREO (50 mg/kg) and (150 mg/kg) groups, the serum levels of inflammatory cytokines were lower than in the NAC group; in the PDREO (150 mg/kg) group, serum levels of inflammatory cytokines were almost reduced to control levels. These inflammatory cytokines are often derived from the damaged liver; therefore, the mRNA levels of some important inflammation-related genes in liver tissues were analyzed. Consistent with the serum levels of inflammatory cytokines, the expression levels of the genes encoding TNF-α, IL-6, IL-1β, NLRP3, iNOS, and COX2 were significantly increased in the liver tissue of the model group, and all drugs effectively antagonized this effect (Fig. 5B). PDREO can reduce the expression of inflammatory genes at the mRNA and protein levels and hence exert anti-inflammatory activity.

Close

Fig. 5

Anti-inflammatory effects of PDREO. (A) Serum levels of inflammatory cytokines. (B) Expression levels of inflammatory genes in liver. Data are expressed as mean ± SD (n = 8). ^##P < 0.01 vs. control group; ^**P < 0.01 vs. model group.

Export to PPT

3.5 PDREO can regulate antioxidant enzymes and relieve hepatic oxidative stress

Oxidative stress caused by excessive production of reactive oxygen species (ROS) plays a central role in the pathology of ALI (Li et al., 2018). To determine the effects of PDREO on LPS/D-GalN-induced oxidative stress in the liver, changes in the antioxidant system were analyzed. As shown in Fig. 6A, LPS/D-GalN exposure resulted in dramatically increased content of MDA (2.628 ± 0.464 mmol/mg protein) in liver homogenate compared with the control group (0.903 ± 0.146 mmol/mg protein). Pre-treatment with NAC effectively reduced the levels of MDA (1.511 ± 0.326 mmol/mg protein) compared with the model group. Surprisingly, both PDREO (50 mg/kg) (0.943 ± 0.239 mmol/mg protein) and (150 mg/kg) (0.820 ± 0.122 mmol/mg protein) almost completely inhibited the increase in MDA levels. Additionally, the toxicity of LPS/D-GalN led to a significant decrease in the content of GSH and the activities of SOD and CAT, resulting in imbalance of the liver antioxidant system. Compared with the model group, NAC and PDREO pre-treatment effectively restored GSH levels and SOD and CAT activities in liver tissue. The qRT-PCR results showed that NAC and PDREO upregulate the gene expression of Gpx4, Sod1, and Cat (Fig. 6B).

Close

Fig. 6

Inhibition of PDREO on oxidative stress induced by LPS/D-GalN. (A) MDA and GSH contents and SOD and CAT activities in liver tissue. (B) Normalized mRNA levels of Gpx4, Sod1, Sod2, and Cat in liver tissue. Data are expressed as mean ± SD (n = 8). ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, ^**P < 0.01 vs. model group.

Export to PPT

3.6 PDREO can alter populations of liver-resident and infiltrating macrophages

Inflammatory cytokines are usually secreted by activated macrophages, but studies have shown that the number of liver-resident F4/80 + macrophages (Kupffer cells) was highly decreased in LPS/D-GalN-induced ALI mice. On the contrary, numerous CD16b + macrophages were infiltrated and induced inflammatory responses (Tacke, 2017; Zigmond et al., 2014). As shown in Fig. 7A, in the present study, the number of F4/80 + cells were dramatically decreased in the model group, but this decrease was significantly inhibited in the NAC and PDREO groups. In contrast, CD11b + cells were massively recruited into the liver of the model group, and this was significantly inhibited by NAC and almost completely blocked by PDREO (50 mg/kg) and (150 mg/kg) (Fig. 7B).

Close

Fig. 7

Effects of PDREO on the number of resident and infiltrating macrophages in the liver. Immunohistochemical staining for (A) F4/80 + and (B) CD11b + cells. Cells were quantified using IPP9.0. Data are expressed as mean ± SD (n = 8). ^##P < 0.01 vs. control group; ^**P < 0.01 vs. model group.

Export to PPT

3.7 PDREO can effectively reduce neutrophil infiltration

Activated macrophages can secrete a variety of chemokines, which can recruit neutrophils into damaged liver tissues, thus aggravating inflammatory reaction and liver injury (Ilyas et al., 2016). Consistent with a previous study (Kusakabe et al., 2021), compared with 0.88 ± 0.99 cells/field in the control group, the number of Ly6G + neutrophils significantly increased to 78.00 ± 12.46 cells/field after challenge with LPS/D-GalN (Fig. 8A, B). After pre-treatment with NAC (300 mg/kg) or PDREO (50 mg/kg) and (150 mg/kg) for 3 days, the number of Ly6G + cells decreased to 37.25 ± 10.69, 19.75 ± 10.51, and 2.13 ± 1.25 cells/field, respectively. Tissue myeloperoxidase (MPO) is a biomarker of neutrophil aggregation in tissues (Rennekampff et al., 1995). Therefore, MPO levels in liver tissues were determined. As depicted in Fig. 8C, the MPO level in the model group was significantly increased, and NAC and PDREO pre-treatment could effectively reduce the MPO level, which was basically consistent with the cell count results.

Close

Fig. 8

Inhibitory effects of PDREO on neutrophil infiltration. (A) Immunohistochemistry of Ly6G + neutrophils. (B) Quantification of the results in (A). (C) MPO activity in liver tissues. Data are expressed as mean ± SD (n = 8). ^#P < 0.05, ^##P < 0.01 vs. control group; ^**P < 0.01 vs. model group.

Export to PPT

3.8 PDREO can effectively prevent liver cell apoptosis

LPS/D-GalN treatment can lead to massive hepatocyte apoptosis, resulting in ALI (Kusakabe et al., 2021). TUNEL staining showed that the apoptosis rate of hepatocytes in the model group reached (35.979 ± 2.928)% (Fig. 9A, B), and the apoptosis rate decreased to (5.446 ± 4.225)% after NAC pre-treatment. PDREO treatment could highly efficiently prevent hepatocyte apoptosis; the apoptosis rate in the PDREO (150 mg/kg) group was (0.019 ± 0.036)%, which was even lower than that of the control group (0.114 ± 0.0.114)%. Caspase 3 is essential for apoptosis, and the appearance of cleaved caspase 3 is a hallmark of apoptosis (Porter and Janicke, 1999). Western blot analysis revealed that LPS/D-GalN injection significantly promoted caspase 3 activation, which was verified by the presence of the cleaved caspase 3 bands in Fig. 9C. Consistent with the TUNEL results, weak cleaved caspase 3 bands were still observed in the NAC group, while no cleaved caspase 3 bands were observed in the PDREO (50 mg/kg) and (150 mg/mL) and control groups. The above data show that PDREO has strong anti-apoptotic activity.

Close

Fig. 9

Inhibitory effects of PDREO on LPS/D-GalN-induced apoptosis in acute liver injury. (A) TUNEL staining of apoptotic cells (FITC and DAPI double positive cells). (B) Quantification of the results in (A). Data are expressed as mean ± SD (n = 8). ^#P < 0.05, ^##P < 0.01 vs. control group; ^**P < 0.01 vs. model group. (C) Western blot analysis of caspase 3 and cleaved caspase 3 (n = 3).

Export to PPT

3.9 PDREO significantly reduced LPS/D-GalN-induced mortality in mice

In the model group, mice began to die 8 h after LPS/D-GalN injection, and all mice were dead about 10 h later (Fig. 10A). The mortality rates in the NAC and PDREO (50 mg/kg) groups were 20% and 10%, respectively, at about 14 h, and no more mice died in the PDREO (50 mg/kg) group after that. However, 72 h after modeling, mice in the NAC group began to die one after another, and the survival rate was only 10% at 120 h. In addition, the appearance and activity of mice in the NAC group changed significantly, which was manifested by dull hair, reduced activity, reduced food intake, and significantly reduced body weight, especially at 72 h (Fig. 10B). The PDREO (50 mg/kg) and (150 mg/kg) groups were generally similar to the control group, except that the state was slightly worse and the body weight decreased within 24 h after modeling (Fig. 10B). Surprisingly, all mice in the PDREO (150 mg/kg) group survived. This result further confirms that PDREO has a significant protective effect on LPS/D-GalN-induced liver injury in mice, indicating great potential for use in the pharmaceutical industry.

Close

Fig. 10

Effects of PDREO on (A) the survival rate and (B) the body weight of mice induced by LPS/D-GalN. Data are expressed as mean ± SD (n = 1–10). ^#P < 0.05, ^##P < 0.01 vs. control group of the corresponding time point.

Export to PPT