R₁-barrigenol—a tea sapogenin—suppresses the lipopolysaccharide-induced inflammatory response in microglial cells

Abstract

Certain natural components are effective against inflammation, a hallmark of neurodegenerative disease progression. Although green tea contains sapogenins, their effects on brain function remain unknown. Herein, we focused on R1-barrigenol (R1B)—a major tea sapogenin—and evaluated its anti-inflammatory effects on mouse microglia. R1B treatment decreased mRNA and protein expression levels of inflammatory cytokines in lipopolysaccharide (LPS)-treated mouse microglia compared with those in LPS-treated control microglia. R1B reduced the mRNA expression of M1-type microglia markers and phosphorylation levels of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha. These results suggest that R1B suppresses LPS-induced nuclear factor κ-light-chain-enhancer of activated B signaling, a regulator of M1-type polarization. Therefore, it is suggested that R1B may reduce inflammatory responses by suppressing M1 polarization-type changes, and that R1B may exhibit preventive and therapeutic effects against neurodegenerative diseases.

Introduction

The prevalence of neurodegenerative diseases is expected to increase annually (Stephenson et al., 2018). The suppression of neuroinflammation, a characteristic of neurodegenerative disease progression (Chen et al., 2016, Rogers et al., 1996), is one of the approaches for the management of degenerative neurologic diseases. Microglia are immunocompetent cells of the central nervous system. Microglia, when overactivated by infection or trauma, release pro-inflammatory cytokines (Colonna and Butovsky, 2017). Therefore, repression of microglial inflammatory responses could be an effective strategy for suppressing neuroinflammation of the central nervous system. Several studies have used lipopolysaccharide (LPS), a ligand of Tolllike receptor 4 (TLR4), to induce inflammation in microglial cells as a model for evaluating neuroinflammation (An et al., 2020, Wen et al., 2017) by TLR4 activation.

Under normal conditions, microglia remain in a quiescent surveillance phenotype termed M0 (Davalos et al., 2005). Resident microglia are polarized to the classically activated M1 type in response to inflammation inducers such as LPS. Microglia that are polarized to the M1 phenotype produce pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF)-α and interleukin (IL)-1β, and express high levels of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO) (Orihuela et al., 2016). Conversely, M2-type microglia are effector cells that suppress the inflammatory immune response and produce anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and TGF-β (Tang and Le, 2016). Microglial M1 polarization affects the development of neurodegenerative diseases. Further evaluation of the mechanisms underlying M1/M2 polarization will provide novel insights and effective targets for controlling neuroinflammation-induced neurodegenerative diseases (Jha et al., 2016, Xu et al., 2015).

Natural compounds, such as flavonoids and glycosides, suppress neuroinflammation in microglia (An et al., 2020). Although astaxanthin regulates microglial M1/M2 polarization via inhibition of nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling (Wen et al., 2017), limited studies have researched the effects of natural compounds on M1/M2 polarization.

Green tea, produced from Camellia sinensis, is consumed globally and can improve brain function and psychopathological symptoms (Mancini et al., 2017); however, its effects are not the result of a single component, and research on the individual effects of the numerous components of green tea is ongoing. Theanine and catechin are effective neuroprotective compounds found in green tea (Deb et al., 2019, Pervin et al., 2018). Green tea also contains saponin, a glycoside of sapogenin, which exerts anti-proliferative effects on carcinoma cells (Yoshikawa et al., 2009) and possesses anti-hyperlipidemic effects (Matsuda et al., 2012). However, there are no reports on the effects of tea saponins and sapogenin on brain function.

Owing to limited research on the analytical methods and biological functions of R1B, a major tea sapogenin, we focused on R1B to evaluate the effects of saponins on brain function. Soybean saponin, a plant saponin, is almost completely unabsorbed by humans; it is converted into aglycones by the intestinal bacteria (Hu et al., 2004). We hypothesized that tea saponins may undergo similar kinetics within the body.

Tea (Hashizume, 1973) and Japanese cheesewood Pittosporum tobira (Oh et al., 2014) contain R1B; however, its distribution within the tea plant is unknown. The only reported biological function of R1B is its antimicrobial activity against periodontopathic bacteria (Oh et al., 2014).

Therefore, in this study, we aimed to develop an analytical method for R1B and evaluate its effects on microglia. Specifically, we investigated the concentration of R1B in C. sinensis and assessed its anti-inflammatory effects on mouse microglia. The findings of this study will serve as a reference for commercial applications of green tea and show its potential for improving brain function in neurodegenerative diseases.

Materials and Methods

Chemicals R1B (purity ≥ 98 %) was obtained from Nagara Science (Gifu, Japan). Antibodies against β-actin (Cat. #4970), IκBα (Cat. #4812), and anti-iNOS (Cat. #13120) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-p-IκBα antibody (Cat. #ab133462) and anti-PPAR-γ antibody (Cat. # ab209350) were obtained from Abcam (Cambridge, UK). LPS from Escherichia coli O26 (Cat. #121-05161) was obtained from FUJIFILM Corporation (Osaka, Japan).

Green tea cultivation Camellia sinensis var. assamica (green tea) samples were used to evaluate the effects of the variety and harvest season on R1B concentration. We selected three major varieties that are widely grown in Japan: Yabukita, Sayamakaori, and Benifuki. All three varieties of green tea were grown in Sayama City, Saitama Prefecture, and the leaves, stems, and flowers were collected during the three crop seasons of April, June, and December. The samples were dried at 50 °C, powdered, stored, and analyzed.

Extraction of R1B R1B was extracted from the plant material through a previously reported method with modifications (Hashizume, 1973). The powdered green tea sample (5 g) was extracted with 80 % CH₃OH (400 mL) at 80 °C and 10 MPa. The crude extract was evaporated, dissolved in 10 % HCl and 50 % CH₃OH (140 mL), incubated at 70 °C for 3 h, added to 20 % KOH and 50 % CH₃OH (100 mL), and incubated at 70 °C for 1 h. This solution was then evaporated, dissolved in water (100 mL), and extracted twice with chloroform (200 mL). The extraction rate with chloroform was 99.1 % for the first extraction and 0.9 % for the second extraction. The chloroform extract was then evaporated and dissolved in methanol for HPLC analysis.

HPLC apparatus and conditions R1B was quantified using an HPLC system (5430 diode array detector; Hitachi High-Technologies, Tokyo, Japan) and an Inertsil ODS-3 column (4.6 × 250 mm, 4 µm; GL Sciences, Tokyo, Japan). The mobile phase comprised acetonitrile/methanol/water (4.5:3:4, v/v/v) at a flow rate of 1.2 mL/min. R1B was detected at 210 nm. Each analysis was performed in triplicate. Quantitative analysis of R1B was performed using the calibration curve method.

R1B was also identified using a liquid chromatograph combined with a mass spectrometer (LCMS-2020; Shimadzu, Kyoto, Japan) and detected using an Inertsil ODS-3 column (250 × 2.1 mm, 3 µm; GL Sciences). The mobile phase comprised methanol/water (8.5:1.5, v/v; 0.1 % acetic acid) at a flow rate of 0.2 mL/min for 30 min. The LCMS-2020 mass spectrometer equipped with electrospray ionization (ESI) ionized the sample. The ESI-MS conditions were as follows: gas temperature, 300 °C; nitrogen drying gas, 15 L/min; negative ion mode (ESI−); and mass range, 150–800 m/z. R1B was detected in scan mode with m/z 505.3.

Cell line and R1B treatment BV2 (a mouse microglia line) was obtained from Elabscience Biotechnology (Wuhan, China). BV2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were cultured under 5 % CO₂ at 37 °C and passaged every 2–3 days.

The cells were simultaneously treated with LPS (100 ng/mL) and R1B (10 or 20 µM) for 24 h to evaluate the potential anti-inflammatory effects of R1B. The concentration of LPS and incubation time were determined as previously described (Castellano et al., 2019).

Briefly, BV2 cells were seeded at 4×10⁵ cells/well in 6-well plates. The following day, cells were treated with 100 ng/mL LPS and 10 or 20 µM R1B for 24 h. After treatment, culture supernatants were subjected to NO and cytokine assays, and cells were subjected to mRNA, protein, and viability analyses.

Measurement of cell viability The viability of BV2 cells after 24 h of LPS and R1B treatment was measured using a Cell Proliferation Assay Kit according to the manufacturer’s protocol (Promega, Madison, WI, USA). The colored reaction solution was quantified by measuring absorbance at 490 nm. The viability of treated cells was calculated as the percentage of controls cells.

Measurement of NO The production of NO induced by LPS was measured using the Griess Reagent System (Cat. #G2930, Promega). The cell culture supernatants were collected after 24 h of LPS and R1B treatment, and NO contained in the cell culture supernatant was evaluated, following the manufacturer’s instructions.

Measurement of inflammation-related cytokine levels The cell culture supernatants were collected after 24 h of LPS and R1B treatment. The levels of TNF-α, IL-6, IL-10, and TGF-β were measured using specific enzyme-linked immunoassay (ELISA) kits (TNF-α, IL-6: eBioscience, San Diego, CA, USA; IL-10, TGF-β: Proteintech, Rosemont, IL, USA), as per the manufacturer’s instructions.

RNA extraction and real-time PCR After 24 h of LPS and R1B treatment, total RNA of BV2 cells was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Isolated RNA was reverse-transcribed using a PrimeScript RT Reagent Kit (Takara Bio, Kyoto, Japan). Real-time PCR was performed using the Thermal Cycler Dice Real Time System III (Takara Bio). The relative mRNA expression was assessed using the 2^−ΔΔCT method. For each sample, a parallel reaction was performed with Gapdh as the internal control. Each reaction was performed in duplicate. The primer sequences used for real-time PCR are based on previous research (Di et al., 2017, Friess et al., 2021, Humbert-Claude et al., 2016, Kim et al., 2011, Niidome et al., 2021, Wang et al., 2019, Yang et al., 2017, Zhao et al., 2017) and are listed in Table 1.

Table 1. List of primers used for real-time PCR analysis.

Target gene	Accession number	Forward primers	Reverse primers
Gapdh	NM_008084.2	5′-AGGTCGGTGTGAACGGATTTG-3′	5′-TGTAGACCATGTAGTTGAGGTCA-3′
Tnf-α	NM_001278601.1	5′-ATGGCCTCCCTCTCATCAGT-3′	5′-CTTGGTGGTTTGCTACGACG-3′
Il-6	NM_031168	5′-TAGTCCTTCCTACCCCAATTTCC-3′	5′-TTGGTCCTTAGCCACTCCTTC-3′
iNOS	NM_010927.4	5′-GCGGAGTGACGGCAAACAT-3′	5′-AGGTCGATGCACAACTGGG-3 ′
Cox-2	NM_011198	5′-ACATCCCTGAGAACCTGCAGT-3′	5′-CCAGGAGGATGGAGTTGTTGT-3′
Il-1β	NM_008361.4	5′-TGACGGACCCCAAAAGATGA-3′	5′-TCTCCACAGCCACAATGAGT-3′
Arg-1	NM_007482.3	5′-GAACACGGCAGTGGCTTTAAC-3′	5′-TGCTTAGCTCTGTCTGCTTTGC-3′
CD206	NM_008625.1	5′-TCTTTGCCTTTCCCAGTCTCC-3′	5′-TGACACCCAGCGGAATTTC-3′
Il-10	NM_010548.2	5′-GCTCTTACTGACTGGCATGAG-3′	5′CGCAGCTCTAGGAGCATGTG-3′
Tgf-β	NM_000660.7	5′-CCCGAAGCGGACTACTATGCT-3′	5′-GTTTTCTCATAGATGGCGTTGTTG-3′

Western blotting BV2 cells were collected 24 h after LPS and R1B treatment. The cells were suspended in radioimmunoprecipitation assay buffer and centrifuged at 15 000 × g for 5 min at 4 °C. Supernatants were boiled for 5 min, and total protein levels were measured using a BCA Protein Assay Kit (Takara Bio). Protein (10 µg) was separated using SDS gel electrophoresis and then electrophoretically transferred onto PVDF membranes (Millipore, Burlington, MA, USA). Membranes with the transferred proteins were incubated at 25 °C for 1 h in blocking buffer with primary antibodies (1:1 000), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:1 000) (Cell Signaling Technology) for 1 h at 25 °C. Finally, the blots were developed using the ImmunoStar Zeta Kit (FUJIFILM Wako Pure Chemical Corporation). Luminescence signals were detected using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and their intensity was quantified using Image Lab Software (Bio-Rad Laboratories). Each protein band signal was normalized to that of β-actin.

Statistical analyses Concentrations of R1B in crops from the third harvest season were analyzed using one-way analysis of variance (ANOVA), followed by the post-hoc Tukey–Kramer test as parametric analyses. Changes in R1B concentration at different harvest times and in plant parts were analyzed by two-way ANOVA. Other experimental data on multi-group comparisons were analyzed using ANOVA, followed by the post-hoc Dunnett test for parametric analyses. Statistical significance was set at p < 0.05. Statistical analyses of experimental data were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA).

Results

Analysis of R1B concentration in C. sinensis We analyzed the R1B concentration (Fig. 1A) in green tea using ultraviolet (UV)-HPLC and LC-MS. A single peak of R1B was obtained using UV-HPLC (Fig. 1B). The mass spectrum of the separated peak was consistent with the molecular weight of R1B (Fig. 1C). Thus, we established a suitable method for analyzing R1B.

Fig. 1

Structure and high-performance liquid chromatography (HPLC) chromatograms of R1B. (A) Chemical structure of R1B, a tea sapogenin derived from Camellia sinensis. (B) Typical HPLC chromatograms of R1B standard, tea leaf, and stem extract. (C) Ion chromatograms and mass spectra of R1B standard and tea flower extracts. Mass spectra are shown for the peaks indicated by arrows.

Variations in R1B concentration in C. sinensis We analyzed the R1B concentration per dry weight of three green tea varieties, Yabukita, Sayamakaori, and Benifuki, based on the harvest time and plant parts, namely leaves, stems, and flowers (Fig. 2).

Fig. 2

R1B concentration per dry weight in C. sinensis (%) (n = 3). Results are displayed as mean ± standard error of mean (SEM), ^*** p < 0.005.

We performed two analyses using the HPLC analysis results. First, to compare the R1B content in each plant part, we performed multiple comparisons of the results of crops from the third harvest against that of the three plant parts (leaves, stems, and flowers). We found that the flowers had a higher R1B concentration than the leaves and stems. (Yabukita: ANOVA: F_(2,6) = 130.8, p < 0.0001, Tukey test: leaves vs flower p < 0.005, stem vs flower p < 0.005; Sayamakaori: ANOVA: F_(2,6) = 84.79, p < 0.0001, Tukey test: leaves vs flower p < 0.005, stem vs flower p < 0.005; Benifuki: ANOVA: F_(2,6) = 707.5, p < 0.0001, Tukey test: leaves vs flower p < 0.005, stem vs flower p < 0.005).

Subsequently, to investigate the effect of harvest time on R1B concentration, we compared its concentrations in stems and leaves using two-way ANOVA (variable factors: section of plant and harvest time). The R1B concentration decreased over time in Sayamakaori and Benifuki (Yabukita: harvest time, ANOVA: F_(2,12) = 2.894, p = 0.09; Sayamakaori: harvest time, ANOVA: F_(2,12) = 106.9, p < 0.0001; Benifuki: harvest time, ANOVA: F_(2,12) = 61.68).

Effects of R1B on viability and release of inflammatory cytokines and NO in LPS-treated cells We analyzed the effect of R1B on the release of inflammatory cytokines induced by LPS in microglia cells. First, we evaluated the effect of various R1B concentrations on BV2 cell viability (Fig. 3A). We confirmed that cell viability was not affected by 10 or 20 µM R1B (ANOVA: F_(3,28) = 2.302, p = 0.098). In addition, LPS increased TNF-α, IL-6, and NO levels, and these increases were suppressed by 20 µM R1B treatment. (TNF-α: ANOVA: F_(3,20) = 874.9, p < 0.0001, Dunnett test: p < 0.005; IL-6: ANOVA: F_(3,20) = 75.99, p < 0.0001, Dunnett test: p < 0.005; NO: ANOVA: F_(3,28) = 289.6, p < 0.0001, Dunnett test: p < 0.005; Fig. 3B, C, D).

Fig. 3

Effects of R1B on inflammatory cytokine and nitric oxide (NO) production.

(A) Effects of R1B and LPS on cell viability (n = 8). (B, C, D) Effects of R1B on the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and NO. (n = 6) Results are displayed as mean ± SEM; ^* p < 0.05, ^**p < 0.01, ^*** p < 0.005 compared with LPS(+)/R1B(-)-treated cells.

Effects of R1B on cytokine- and iNOS-related mRNA and protein expression We analyzed the mRNA expression of inflammatory cytokines and iNOS. LPS increased the expression of TNF-α and IL-6 mRNA, and this increase was suppressed by treatment with 20 µM R1B (TNF-α: ANOVA: F_(3,20) = 642.0, p < 0.0001, Dunnett test: p < 0.0001; IL-6: ANOVA: F_(3,20) = 305.8, p < 0.0001, Dunnett test: p < 0.005; Fig. 4A, B). Although LPS increased the mRNA expression of iNOS, treatment with 20 µM R1B did not affect iNOS expression (ANOVA: F_(3,20) = 292.4, p < 0.0001, Dunnett test: p = 0.99; Fig. 4C). In contrast, the LPS-induced increase in iNOS protein expression was decreased by treatment with 10 µM R1B (ANOVA: F_(2,12) = 23.65, p < 0.0001, Dunnett test: p < 0.01; Fig. 4D, E).

Fig. 4

Effects of R1B on cytokine and NO-related mRNA expression. (A, B) mRNA expression of inflammatory cytokines TNF-α and IL-6 (n = 6). (C) Inducible nitric oxide synthase (iNOS) mRNA expression level (n = 6). (D, E) Protein expression of iNOS. Results are displayed as mean ± SEM (n = 5); ^*p < 0.05, ^** p < 0.01, ^*** p < 0.005 compared with LPS(+)/R1B(-)-treated cells.

Effects of R1B on microglial polarization We analyzed the effects of R1B on microglial polarization. Levels of M1 markers cyclooxygenase-2 (COX-2) and IL-1β increased with LPS treatment; however, this increase was suppressed by R1B treatment (COX-2: ANOVA: F_(2,15) = 451.3, p < 0.0001, Dunnett test: p < 0.005; IL-1β: ANOVA: F_(2,15) = 1455, p < 0.0001, Dunnett test: p < 0.005; Fig. 5A, B). Levels of M2 markers arginase-1 (Arg-1) and CD206 decreased with LPS treatment and were not significantly affected by treatment with 20 µM R1B (Arg-1: ANOVA: F_(2,15) = 625.1, p < 0.0001, Dunnett test: p = 0.075; CD206: ANOVA: F_(2,15) = 131.8, p < 0.0001, Dunnett test: p = 0.285; Fig. 5C, D). In contrast, levels of secretory M2 markers IL-10 and TGF-β increased by treatment with 20 µM R1B compared to that in LPS(+)/R1B(-)-treated cells (IL-10: ANOVA: F_(2,15) = 25.17, p < 0.0001, Dunnett test: p < 0.005; TGF-β: ANOVA: F_(2,15) = 138.6, p < 0.0001, Dunnett test: p < 0.05; Fig. 5E, F).

Fig. 5

Effects of R1B on the expression of M1 and M2 mRNA markers. (A, B) Expression of M1 markers cyclooxygenase-2 (COX-2) and IL-1β (n = 6). (C–F) Expression of M2 markers arginase-1 (Arg-1), CD206, IL-10, and TGF-β (n = 6). Results are displayed as mean ± SEM; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.005, compared with LPS(+)/R1B(−)-treated cells.

Furthermore, we analyzed the effect of R1B on protein levels of the M2 markers IL-10 and TGF-β. IL-10 protein expression was not affected by treatment with LPS or 20 µM R1B (ANOVA: F_(2,21) = 0.7942, p = 0.465; Fig. 6A). Similarly, TGF-β protein expression was not affected by LPS; however, treatment with 20 µM R1B decreased TGF-β protein expression compared to that in LPS(+)/R1B(-)-treated cells (ANOVA: F_(2,21) = 8.805, p = 0.0017, Dunnett test: p < 0.016; Fig. 6B).

Fig. 6

Effects of R1B on the protein expression of M2 mRNA markers. Expression of M2 markers IL-10 and TGF-β (n = 8). Results are displayed as mean ± SEM; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.005, compared with LPS(+)/R1B(−)-treated cells.

Effects of R1B on NF-κB and PPAR-γ signaling We analyzed the effect of R1B on phosphorylated (p)-IκBα protein level and found that treatment with 20 µM R1B decreased the LPS-induced increase in p-IκBα concentration (ANOVA: F_(2,12) = 13.27, p < 0.001, Dunnett test: p < 0.05; Fig. 7A, B). Furthermore, we analyzed the effect of R1B on PPAR-γ protein level and found that both LPS and 20 µM R1B treatment did not affect PPAR-γ protein level (ANOVA: F_(2,12) = 0.9564, p < 0.4117; Fig. 7C, D)

Fig. 7

Effects of R1B on p-IκBα and PPAR-γ protein level. (A) p-IκBα protein levels were determined by western blotting. (B) Quantitative analysis of p-IκBα/IκBα levels. (n = 5) (C) PPAR- γ protein levels were determined by western blotting. (D) Quantitative analysis of PPAR- γ/β-actin levels (n = 5). Results are displayed as mean ± SEM; ^* p < 0.05, ^** p < 0.01, compared with LPS(+)/R1B(−)-treated cells.

Discussion

In this study, we analyzed the R1B content in C. sinensis and evaluated its effects on inflammation in microglial cells. Our results showed that R1B is abundant in the flowers of C. sinensis, and it substantially suppresses neuroinflammation via NF-κB signaling.

We developed an analytical method for R1B and investigated its concentration in typical green tea varieties grown in Japan (Yabukita, Sayamakaori, and Benifuki) based on harvest time and plant parts. We found that the flowers contained a higher quantity of R1B than the leaves and stems. Sapogenin exists in the plant body as saponin bound to a sugar molecule. The saponins in green tea are found in different parts of the plant. For example, floratheasaponins A–C are found in flowers (Yoshikawa et al., 2005), assamsaponin I is found in seeds, and assamsaponin J is found in leaves (Murakami et al., 2000). These previous studies suggest that the R1B concentration may also vary by location in the plant, which is consistent with the results of this study. Moreover, our result that R1B is present in tea flowers is consistent with a previous study (Matsuda et al., 2016). Furthermore, our results provide new evidence of the presence of R1B in the tea leaves and stems.

It was observed that R1B concentration in the leaves and stems was higher in plants harvested earlier in the year. Saponins are a part of the plant defense system, and some saponins themselves possess antimicrobial effects or can activate plant immunity (Trdá et al., 2019). An elevated R1B concentration in the early harvest period may represent a plant defense mechanism, wherein an elevated saponin concentration is observed during the new leaf stage. This is the first report on sapogenin concentration in tea plants during different harvest periods, which could contribute to practical applications by enabling the selection of the time of harvest when R1B concentration is at its highest.

Yabukita and Sayamakaori constitute the major green tea varieties in Japan. The R1B concentration in Benifuki was higher than that in Yabukita or Sayamakaori, suggesting that the saponin concentration may vary according to species/variety. However, it should be noted that we did not monitor the growing conditions of tea plants. Thus, clarification of the R1B content in relation to growing conditions, such as temperature and precipitation, would contribute to further understanding of the physiological significance of saponins in plants and their commercial use.

Neuroinflammation is associated with a decreased number of neurons and dendrites, suggesting that it may be an etiological factor in neurodegenerative diseases (Stephenson et al., 2018). Microglial activation is associated with neurodegenerative diseases and is an early feature of Alzheimer’s disease (Heneka et al., 2015). Thus, the regulation of microglial activation can contribute to the prevention and treatment of neurodegenerative diseases (Subhramanyam et al., 2019).

In this study, we found that R1B suppressed the LPS-induced inflammatory response in microglia in vitro by decreasing the levels of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6) and NO. As R1B is classified as a triterpene, this result is consistent with the results for other triterpenes; for example, triterpenes from the rice paper plant Tetrapanax papyrifer and oleanolic acid (Castellano et al., 2019, Cho et al., 2016). R1B also decreased TNF-α and IL-6 mRNA levels in LPS-stimulated microglia, whereas the levels of iNOS, an NO synthase, were unaffected. In contrast, iNOS protein expression decreased with R1B treatment, suggesting that R1B may regulate iNOS expression post-transcriptionally. The post-transcriptional regulation of iNOS gene expression is achieved by controlling the stability of iNOS mRNA (Kiemer and Vollmar, 1998) and the catalytic activity (Kunz et al., 1996) and stability of the protein (Won et al., 2004). The effects of R1B on iNOS mRNA and protein stability remain unknown; thus, further analysis of protein stability and catalytic activity of iNOS as a result of R1B treatment is required.

In this study, levels of COX-2 and IL-1β, which are markers of M1 microglia, decreased with R1B treatment compared with those in LPS-stimulated microglia, and levels of Arg-1 and CD206, which are markers of M2 microglia, did not increase. These results are consistent with previous studies that demonstrated that M1 microglia rarely switch to the M2 phenotype once activated (Orhuela et al., 2016).

IL-10 and TGF-β mRNA expression was upregulated by LPS treatment and further upregulated with R1B treatment. In contrast, LPS treatment did not increase IL-10 and TGF-β protein expression. In previous studies, the IL-10 mRNA expression of microglia decreased following 4 h of LPS treatment (Wen et al., 2017), whereas treatment for 10 h did not affect IL-10 protein expression (Li et al., 2020b). Similarly, IL-10 protein production was not affected by 12 h of LPS treatment (1000 ng/mL); however, its expression level increased at 24 h and further increased at 48 h (Sun et al., 2019). Thus, LPS dose and treatment time may affect the release of IL-10 and TGF-β. In this study, 24 h LPS treatment did not affect IL-10 and TGF-β protein release; however, prolonging treatment time may increase protein release. Therefore, further research on the effects of LPS dose and treatment time on inflammation-related cytokine levels is required.

R1B reduced the levels of M1 microglial markers, suggesting that R1B may suppress M1 polarization, thus suppressing inflammatory responses. To further interpret this, we elucidated the mechanism by which R1B suppresses M1 polarization.

NF-κB activity is maintained at a high level in neurotoxic M1 microglia but is downregulated in neuroprotective M2 microglia. NF-κB is a regulatory transcription factor involved in microglial polarization (Ni et al., 2015). As NF-κB is activated by proteolysis and the nuclear translocation of IκBα, IκBα is a key regulator of NF-κB signaling. Under normal conditions, NF-κB signaling is not upregulated, and IκBα and NF-κB form a complex outside the nucleus. Phosphorylation of IκBα leads to the degradation of IκBα, resulting in NF-κB activation (Li et al., 2020a).

Based on this, we hypothesized that R1B suppresses NF-κB signaling through IκBα and represses M1 polarization; hence, we evaluated the effect of R1B on IκBα. The results showed that R1B decreased p-IκBα levels, suggesting that R1B suppresses M1 polarization and the LPS-induced inflammatory response by repressing IκBα phosphorylation and NF-κB signaling.

In contrast, the activation of PPAR-γ is a characteristic of M2 polarization (Orhuela et al., 2016). R1B did not affect the mRNA expression of M2 marker genes in LPS-treated cells, suggesting that R1B may not affect M2 polarization in the presence of LPS. To confirm this, we analyzed the protein expression of the M2 marker gene PPAR-γ. The results showed that R1B did not affect PPAR-γ protein expression. This is consistent with the results of the M2 marker gene expression analysis.

Our study has certain limitations. The cultivation trials in the present study were conducted from 2020 to 2021; thus, investigations over longer periods of time are required to gain a better understanding of R1B concentration in tea. Suppression of NF-κB signaling is only one of the mechanisms whereby R1B suppresses inflammation. Comprehensive gene expression analysis is needed to elucidate the mechanism whereby R1B suppresses LPS-induced inflammation. The ability of R1B to penetrate the blood-brain barrier is unknown, and it is unclear whether oral intake of R1B will induce a neuroprotective effect in animals and humans. In the future, the pharmacokinetics of R1B in animals and humans, and whether R1B improves cognitive function by suppressing microglial inflammation should be evaluated.

In summary, we focused on R1B in green tea and determined its concentration in three varieties based on plant parts and harvest season. We further showed that R1B suppressed the LPS-induced inflammatory response in microglia by inhibiting M1 polarization through the suppression of NF-κB signaling.

This study is significant because it is the first report on the concentration and biological functions of R1B in green tea. This study showed that R1B may be effective in alleviating inflammation in neurodegenerative diseases. The effect of R1B in C. sinensis should be studied further in animals and humans to improve the health and quality of life of people with neurodegenerative diseases.

Acknowledgements This study was funded by the Nippn Corporation. We thank Ms. Akiko Yuzurihara for her assistance with the extraction of R1B, Mr. Takahiro Yokota for providing the tea samples, and Editage (www.editage.com) for English language editing.

Author Contributions R.H., S.F., K.A., and S.K. designed the experiments. R.H. performed the experiments and analyzed the data. R.H. and S.K. wrote the manuscript.

Conflict of interest R.H., S.F., and K.A. are employed by Nippn Corporation. The other author declares no conflicts of interest.

Abbreviations

ANOVA

analysis of variance

Arg-1

arginase-1

COX-2

cyclooxygenase-2

ESI

electrospray ionization

HPLC

high-performance liquid chromatography

IκBα

nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor

IL

interleukin

iNOS

inducible nitric oxide synthase

LC-MS

liquid chromatography-mass spectrometry

LPS

lipopolysaccharide

NF-κB

nuclear factor κ-light-chain-enhancer of activated B cells

NO

nitric oxide

PPAR-γ

peroxisome proliferator-activated receptor gamma

R1B

R₁-barrigenol

TGF-β

transforming growth factor-β

TNF-α

tumor necrosis factor-α

UV

ultraviolet

References

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