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Protective effect of Codonopsis lanceolata root extract against alcoholic fatty liver in the rat
J Med Food
Abstract
Alcohol intake remains the most important cause of fatty liver throughout the world. The current study was undertaken to determine whether dietary supplementation with Codonopsis lanceolata root water extract attenuates the development of alcoholic fatty liver in rats and to elucidate the molecular mechanism for such an effect. Male Sprague-Dawley rats were fed normal diet (ND), ethanol diet (ED) (36% of total energy from ethanol), or 0.5% C. lanceolata root extract-supplemented ethanol diet (ED+C) for 8 weeks. C. lanceolata root water extract supplemented to rats with chronic alcohol consumption ameliorated the ethanol-induced accumulations of hepatic cholesterol and triglyceride. Chronic alcohol consumption up-regulated the hepatic expression of genes involved in inflammation, fatty acid synthesis, and cholesterol metabolism, including tumor necrosis factor alpha (TNFalpha), liver X receptor alpha (LXRalpha), sterol regulatory element-binding protein (SREBP)-1c, fatty acid synthase, acetyl-coenzyme A carboxylase alpha (ACC), stearoyl-coenzyme A desaturase 1, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), and low-density lipoprotein receptor (LDLR). The ethanol-induced up-regulations of TNFalpha, LXRalpha, SREBP-1c, HMGR, and LDLR genes in the liver were reversed by feeding C. lanceolata root water extract for 8 weeks. Moreover, ethanol-induced decreases in the ratio of phospho-5′-AMP-activated protein kinase (AMPK) alpha/AMPKalpha and phospho-ACC/ACC protein levels in the liver were significantly restored (135% and 35% increases, respectively, P < .05) by supplementing them with C. lanceolata root water extract. In conclusion, C. lanceolata root water extract appears to be protective against alcoholic fatty liver through the regulation of SREBP-1c, LXRalpha, HMGR, and LDLR genes and by the phosphorylation of AMPKalpha and ACC, which are implicated in lipid metabolism.
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Received26 Jun 2021
Revised13 Jul 2021
Accepted27 Jan 2022
Codonopsis lanceolata is a perennial smelly herbaceous plant and widely employed for the treatment of various lung cancer and inflammation. However, the anticancer substances in C. lanceolata and their underlying mechanisms had not been well clarified. In this study, six compounds were obtained from the water extracts of C. lanceolata polyacetylenes (CLP) and then identified as syringin, codonopilodiynoside A, lobetyol, isolariciresinol, lobetyolin, and atractylenolide III. Treatment with CLP remarkably suppressed the cell proliferation, colony formation, migration, and invasion of A549 cells. Synergistic effects of lobetyolin and lobetyol were equivalent to the antiproliferative activities of CLP, while other compounds did not have any inhibition on the viabilities of A549 cells. CLP also reduced the expression of Ras, PI3K, p-AKT, Bcl-2, cyclin D1, and CDK4 but increased the expression of Bax, GSK-3β, clv-caspase-3, and clv-caspase-9, which could be reversed by the PI3K activator 740YP. Furthermore, CLP retarded the growths of tumor and lung pathogenic bacteria in mice. It demonstrated that lobetyolin and lobetyol were the main antitumor compounds in C. lanceolata. CLP induced cell apoptosis of lung cancer cells via inactivation of the Ras/PI3K/AKT pathway and ameliorated lung dysbiosis, suggesting the therapeutic potentials for treating human lung cancer.
Introduction
Fetal bovine serum (FBS) was acquired from Tianhang Biotech. Co. Ltd., Hangzhou, China. MTT was obtained from Sigma-Aldrich (China). Antibodies used in the study were obtained from Cell Signaling Technology (Danvers, USA) or Abcam (Cambridge, UK). Other regents were all purchased from Hangzhou Bozan Biotech. Co. Ltd., China.
Extraction and Isolation of Compounds in CLP
The cells were seeded into the 96-well plates (each well had 5000 cells) and then treated with CLP (2.5, 5, and 10 μg/mL) or DDP (5 μg/mL) for 24, 48, and 72 h. Twenty microliters of PBS solution containing 5 mg/mL of MTT was assigned to each well and incubated at 37°C for 4 h. At last, each well was mixed with 150 μL of dimethyl sulfoxide. The absorbance was measured at 490 nm.
Cells were seeded into six-well plates (103 cells/well) and then treated with CLP (2.5, 5, and 10 μg/mL) or DDP (5 μg/mL). When a clearly visible colony appeared in the culture dish, the cells were fixated with methanol and subsequently dyed with 10% Giemsa for 10–30 min. Colonies were counted under an optical microscope (OLYMPUS, Japan).
Transwell Invasion and Wound Healing Assays
The serum-free medium containing cells were added into the upper chambers of the transwell chambers (8 μm pore size) with Matrigel (BD Biosciences, USA), while 500 μL of 20% FBS medium was presented into the matched lower chambers. Forty-eight hours after incubation with test drugs, only the lower chambers were collected, fixated with methanol for 30 min, and stained with 0.1% crystal violet for 15–30 min. The cells on the lower surfaces of the chambers were counted under the optical microscope (OLYMPUS, Japan).
The cells were seeded into six-well plates (each well had cells). The cell layer of each well was wounded by using the tip of a 200 μL pipette. The wells were carefully washed with PBS to remove the detached cells, and then, the remaining cells were cultured at 37°C for 48 h. Images were captured at 0 and 48 h after scratching, and the wound width in each well was measured with a ruler under the microscope (OLYMPUS, Japan).
Cell Apoptosis and Cell Cycle Assay
The cells in 6-well plates were treated with CLP at the concentrations of 2.5–10 μg/mL for 24 h and then mixed with 500 μL of buffer, 5 μL of annexin V FITC (20 μg/mL), and 10 μL of PI (50 μg/mL). The apoptotic rates of CLP-treated A549 cells were detected by using flow cytometry (BD, USA). On the other hand, the cells were also collected for examining cell cycle distribution according to the commercial kit (MultiSciences Biotech Co. Ltd., Hangzhou, China). Cells were treated with 500 μL of buffer and 5 μL of permeabilization solution and then kept for 20 min at room temperature and no-light conditions. Finally, the cell cycles of stained cells were assayed by flow cytometry.
Western Blot Assay
The total proteins of cells or tumor samples were extracted with 0.2 mL of RIPA, 1 μL of PMSF, and 1 μL of the phosphorylation protease inhibitor. Then, the supernatant of the protein extracts was collected and its quality was controlled by the BCA detection kits (KeyGEN BioTECH Co. Ltd., Nanjing, China). The total proteins were diffused on 12% SDS-PAGE electrophoresis and transferred onto the polyvinylidene difluoride membranes. These membranes were soaked in 5% nonfat milk for 2 h and then treated with primary antibodies for 10 h at 4°C. After pretreatment with TBST for 3 times, the membranes were treated with secondary antibodies for 1 h. The expression of the target proteins was measured by using chemiluminescence (Beyotime, China). GAPDH was considered as the control for Western blot analysis.
Animals and Experimental Procedure
The growth of orthotopic tumor was monitored every 5 days by using the IVIS Lumina LT imaging system (PerkinElmer, USA). Briefly, the mice were anesthetized by isoflurane and then intravenously injected with 1.5 mg D-luciferin (Yeasen, China) 10 min prior to imaging.
HE and TUNEL Assays
Tumor tissues were prepared as in Section 2.9. The paraffin-embedded samples excised from A549 nude mice were stained by using Ki-67 and pAKT antibodies for immunohistochemistry. Images of the tumor tissues were captured using a light microscope (Leica DM2500, Germany).
Bacterial 16S rDNA Sequencing
Each assay was performed 3 times. The data were showed as the deviation (SD). Statistical differences were analyzed by one-way ANOVA by using the GraphPad Prism 6 and SPSS 16.0 software. The significant differences between 2 groups were set at
Results
To control the quality of the herbal extract CLP, we determined the contents of the six compounds by HPLC. The representative HPLC chromatograph was presented as shown in Figure 1(a). The contents of syringin, codonopilodiynoside A, lobetyol, (+)-isolariciresinol, lobetyolin, and atractylenolide III in CLP were 46.9%, 5.7%, 9.3%, 12.6%, 10.5%, and 1.1%, respectively.
Effect of CLP on A549 Cell Proliferation
To determine the anticancer pharmacodynamic substances in CLP, the six compounds isolated from CLP were investigated in vitro in A549 cell model. As shown in Figure 1(d), lobetyol, lobetyolin, and atractylenolide III had significant inhibition on the cell proliferation of A549 cells, while other compounds showed little or no effects. Considering that the contents of those six compounds in CLP were clear, the contribution of each compound to the antiproliferative activities of CLP could be calculated by comparing their overall and individual inhibition. The inhibitory rates of CLP (20 μg/mL), syringin (9.38 μg/mL), lobetyol (1.86 μg/mL), lobetyolin (2.10 μg/mL), and atractylenolide III (0.22 μg/mL) on the proliferation of A549 cells were 65%, 8.1%, 21.8%, 24.4%, and 2.3%, respectively. Therefore, lobetyol and lobetyolin contributed approximately 71% to the inhibitory effects of CLP on lung cancer cell proliferation (Figure 1(e)). Although the content of syringin in CLP was approximately fivefold higher than those of lobetyol and lobetyolin, the antiproliferative activity of syringin was approximately fourfold less. Thus, lobetyol and lobetyolin could be the main anticancer compounds. Moreover, the combination index of lobetyol and lobetyolin, which were mixed at 5 different concentrations (Figure 1(f)), was around the additive baseline 1, indicating their additive effects.
Effects of CLP on A549 Cell Migration, Invasion, and Colony Formation
As shown in Figure 2, the width of wound scratch in the control group was significantly reduced 48 h after CLP treatment. However, compared with the untreated group, wound closures were significantly decreased in the CLP-treated groups ( indicating the inhibition of CLP on A549 cell migration. Moreover, the results of transwell assay showed that approximately 250 cells invaded the lower chamber in the control group after CLP treatment for 48 h. However, the number of cells in the lower chamber was significantly decreased in the CLP-treated groups compared with the control group ( indicating the inhibition of CLP on A549 cell invasion. Similarly, CLP dose dependently inhibited the colony formation of A549 cells. But colonies were hardly found in 10 μg/mL of the CLP-exposed group.
Effects of CLP on A549 Cell Apoptosis
As shown in Figure 3, the apoptosis rates of the normal cells were only 5% but it was significantly increased in the CLP-treated groups in a concentration-dependent manner ( indicating that CLP induced A549 cell apoptosis. Furthermore, the number of untreated A549 cells at the G1 phase was 67%, which was much lower than those of CLP-treated groups ( In other words, CLP obviously caused an accumulation of A549 cells at the G1 phase and decreased in the S phase in a concentration-dependent manner (
Effects of CLP on the Expression of Ras/PI3K/AKT Signals
At 48 h after CLP treatment, the expression levels of Ras, PI3K, AKT, and pAKT were measured by Western blot analysis. In Figure 4, CLP significantly inhibited the expression levels of Ras, PI3K, AKT, and pAKT compared with the control group ( However, there were no significant differences of PTEN expression among the control and CLP-treated groups ( Therefore, the proapoptotic effect of CLP on lung cancer cells did not depend on the activation of PTEN. Furthermore, after CLP treatment, the expression of Bcl-2, caspase-9, and caspase-3 was significantly reduced but the levels of Bax, clv-caspase 9, and clv-caspase 3 were significantly increased with the increase of CLP concentration (
Since the results of flow cytometry assay showed the cell cycle arrest at the G1 phase induced by CLP, the effect of CLP on the expression of cyclin D1 and CDK4, which were critical for the G1/S transition, was further examined. The results in Figure 4 displayed that the expression of cyclin D1 and CDK4 in the CLP-treated groups was significantly decreased compared with that in the control group ( supporting the G1/S arrest of cell cycle exposed by CLP. Notably, the expression of GSK-3β, which was related to stabilization of cyclin D1, was significantly upregulated after CLP treatment, indicating that CLP arrested A549 cells at the G1 phase via mediating the GSK-3β/cyclin D1/CDK4 pathway.
Antitumor Effects of CLP In Vivo
The growth of orthotopic tumor was monitored using the IVIS Lumina LT imaging system. Tumor volumes and weights were represented by radiance. As shown in Figure 5, the tumor growth of the model group was very fast, especially on the 17th day after challenge. As shown in Figure 5, the volumes of tumor in the model group were much higher than the lung. However, both tumor volumes and weights in the CLP-treated groups were much less than those in the model group ( indicating that CLP could effectively inhibit tumor growth after 15 days of treatment. Moreover, the tumor volumes and weights in the CLP-H group were significantly decreased compared with those in the DDP group ( indicating the stronger potential of CLP on the inhibition of tumor growth in vivo. Interestingly, no significant change was observed in the body weights of tumor-bearing mice between the model and DDP group, while the mice in the CLP-treated group gained more weights than those in the model group ( Therefore, CLP would be less toxic to the mice than DDP.
TUNEL staining was used to visualize the cell apoptosis in tumor tissue. As shown in Figure 6, the apoptotic cells (marked as green) hardly existed in the model group, while tumor tissue from the CLP-treated group exhibited a higher percentage of apoptotic cells compared with that from the model group. Therefore, CLP inhibited tumor growth by the augmentation of apoptotic tumor cells.
The tumor cells characterized with markedly large nuclei were aligned tightly and irregularly in the tissues of the model group. However, after CLP treatment, the adhesion of human lung adenocarcinoma cells disappeared and separated from the surrounding cells, the cell volume was reduced, and the nucleoplasm was condensed. In addition, the immunohistochemistry results displayed that the tumor tissues in the CLP-treated groups presented the low brown expression of Ki-67, an antigen indicating the proliferative state of active tumor cells, while those in the tumor tissues of the model group were comparatively high (Figure 7). Similarly, the expressing profiles of pAKT in the tumor tissues were consistent with those of Ki-67. All these results demonstrate that CLP effectively inhibited the growth of A549 cells in vivo.
Antibacterial Activity of CLP
In Figure 8, the community richness among the model and CLP-treated groups was considered to assess the effects of CLP on lung dysbiosis in A549 tumor-bearing mice. The levels of 3 indexes (ACE, Chao1, and Shannon), which reflected the microbiota diversities, were significantly increased in the CLP-treated groups as the dose increased compared with those in the model group ( The levels of Simpson in CLP-treated groups were decreased compared with those in the model group, but the Simpson levels were significantly different among the CLP-treated groups. Furthermore, the relative abundances of microbes in Veillonella, Streptococcus, and Megasphaera families in the lung tissues of A549 tumor-bearing mice were much higher than those in the CLP-treated mice ( while the relative abundances of Alloprevotella and Actinomyces in the model mice were remarkably lower than those in the CLP-treated mice ( Therefore, CLP improved the lung dysbiosis of the mice with lung cancer.
CLP significantly inhibited the growth of S. pyogenes and S. aureus with MIC values of 1.94 and 2.37 mg/mL and MBC of 1.94 and 4.74 mg/mL. Furthermore, the diameters of bacteriostatic zones of CLP were 12 and 7 mm. These results suggested the potential antibacterial activities of CLP, which would contribute to its regulation on lung dysbiosis induced by cancer cells.
Discussion
In summary, CLP inhibited proliferation and induced apoptosis of A549 cells, which were arrested at the G1/S phase, and suppressed growth of lung cancer in the nude mouse xenograft models. It also significantly upregulated the expression of Bax, GSK-3β, clv-caspase-9, and clv-caspase-3 and downregulated the expression of Ras, Pi3K, pAKT, cyclin D1, CDK4, Ras, Bcl-2, caspase-9, and caspase-3 in A549 cells, which all were reversed by the PI3K activator. But CLP hardly altered the expression of PTEN. Thus, it indicated that CLP induced apoptosis of A549 cells via regulating the Ras/PI3K/AKT pathway. Moreover, CLP exerted antibacterial activities in vitro and improved the lung dysbiosis of tumor-bearing mice. It could be a therapeutic candidate for the prevention and treatment of human lung cancer.
Data Availability
The data is available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
This work is supported by the Zhejiang Science and Technology special fund for research institutes (no. 2015F50065) and Innovation Discipline Construction of Laboratory Animal Genetic Engineering (no. 201604).
