2024 年 47 巻 1 号 p. 138-144
Sjögren’s syndrome (SS) is an autoimmune disorder characterized by oral dryness that is primarily attributed to tumor necrosis factor alpha (TNF-α)-mediated reduction in saliva production. In traditional Chinese medicine, goji berries are recognized for their hydrating effect and are considered suitable to address oral dryness associated with Yin deficiency. In the present study, we used goji berry juice (GBJ) to investigate the potential preventive effect of goji berries on oral dryness caused by SS. Pretreatment of human salivary gland cells with GBJ effectively prevented the decrease in aquaporin-5 (AQP-5) mRNA and protein levels induced by TNF-α. GBJ also inhibited histone H4 deacetylation and suppressed the generation of intracellular reactive oxygen species (ROS). Furthermore, GBJ pretreatment reserved mitochondrial membrane potential and suppressed the upregulation of Bax and caspase-3, indicating that GBJ exerted an antiapoptotic effect. These findings suggest that GBJ provides protection against TNF-α in human salivary gland cells and prevents the reduction of AQP-5 expression on the cell membrane. Altogether, these results highlight the potential role of GBJ in preventing oral dryness caused by SS.
Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by impaired salivary gland function, which can lead to progressive xerostomia.1–3) The role of tumor necrosis factor-alpha (TNF-α), which is closely associated with salivary gland dysfunction and inflammation in SS, in inducing apoptosis in salivary gland has been reported.4,5) Decreased mitochondrial membrane potential is a contributor to apoptosis, and several apoptosis-related proteins, including caspase-3, Bcl-2, and Bax, are involved in this process.6,7)
Aquaporin-5 (AQP-5) is a water channel protein primarily involved in water movement and responds to osmotic or hydrostatic pressure gradients and facilitates the rapid movement of cellular water.8) TNF-α was found to reduce AQP-5 expression and impair AQP-5-mediated water transport.9–11) AQP-5 plays a crucial role in saliva secretion, and TNF-α is considered to lead to a reduction in AQP-5 expression through the increased intracellular accumulation of reactive oxygen species (ROS) and histone H4 acetylation.12,13)
In traditional Chinese medicine, the pathogenesis of SS is related to a deficiency in “Yin” or the body’s fluid balance and that herbal medicines can aid in improving Yin deficiency. Goji berries are the fruits of the deciduous shrub Lycium barbarum L., which belongs to the Solanaceae family and is native to northwest China. Goji berries, which are known for their nourishing Yin properties. Goji berries exhibit a wide range of biological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects. Goji berries are renowned for their abundant composition, specifically their elevated levels of carotenoids, which contribute to their nutritional and bioactive properties.14–16) Due to these beneficial effects and Goji berries have been utilized as an essential dietary component to promote health and well-being in daily life for centuries.
In the present study, we aimed to evaluate the preventive effects of Goji berries on oral dryness in individuals with SS. Specifically, we determined whether pretreatment with goji berry juice (GBJ) prevented TNF-α-induced oral dryness in an in vitro model using human salivary gland cells.
A total of 500 mL GBJ (100% fruit juice, Iskra Industry, Japan) was freeze-dried, and the resulting solid was immersed in 40 mL of n-hexane/acetone (3 : 2, v/v) for extraction, followed by 10 min of sonication. Next, the solvent was evaporated using an evaporator (EYELA) to obtain the GBJ powder, which was dissolved in dimethyl sulfoxide (DMSO), sonicated in a warm water bath for 5 min, and centrifuged at 1000 × g for 5 min to obtain a clear supernatant. The sample solutions at concentrations of 1 and 10 mg/mL were prepared for use in experiments. In parallel, an extract of Coptidis Rhizoma, primarily known for its heat-clearing properties with no known nourishing effect as a Yin herb, was prepared using the same method to be used as negative control. We filtered and sterilized all prepared sample solutions (GBJ as GB sample solution and Coptidis rhizome as CR sample solution) using a 0.22 µm Millipore filter (Merck, Darmstadt, Germany) and stored them at −20 °C until use in experiments.
Cell CultureA253 cells, a human salivary gland line (HTB-41; ATCC, Manassas, VA, U.S.A.), were cultured in McCoy’s 5A modified medium (Invitrogen, Thermo Fisher Scientific K.K., Japan, Tokyo, Japan) supplemented with 10% (v/v) endotoxin-free fetal bovine serum (Biosera, Cholet, France) and 1% (v/v) antibiotic solution (10000 units penicillin and 10 mg/mL streptomycin) (Sigma-Aldrich, St. Louis, MO, U.S.A.). We seeded cells in a 6-cm dish at a density of 3.0 × 106 cells and then incubated at 37 °C under a humidified atmosphere of 5% CO2. The cells were passaged thrice a week.
Cell Viability DeterminationThe cell viability of A253 cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay. The experimental procedures were conducted following the protocol.
Inhibition of AQP-5 by TNF-α and Pretreatment with GBJA253 cells were seeded at a density of 1.0 × 106 cells/well onto 6-well tissue culture plates and serum-starved for 12 h in a serum-free medium. The cultures were preincubated with 1 or 10 µg/mL GB sample solution in serum-free medium for 2 h, and human TNF-α (100 units/mL) was added to the medium for an additional incubation for 24 h. At least three technical and biological replicates were conducted.
RT-PCRTotal RNA was extracted using the RNeasy Plus mini kit (Qiagen) and cDNA was synthesized using 1 µg of total RNA of each sample using the ReverTra Ace Quantitative PCR (qPCR) Master Mix kit (Toyobo, Osaka, Japan). The synthesized cDNA was then amplified using the following primers and the Green Master Mix (Promega, Madison, WI, U.S.A.):
The PCR conditions were as follows: 2 min at 95 °C, 30 s at 95 °C, 1 min at 54 °C, and 1 min at 72 °C. The number of cycles was optimized for each primer pair and the final products were incubated for 5 min at 72 °C. The PCR products were separated on a 1.5% agarose gel, and the bands were visualized using ethidium bromide staining under UV transillumination.
Western BlottingAfter removing the cells from the 6-well tissue culture plates, cell membrane and cytoplasmic fractions were obtained using radio immunoprecipitation assay (RIPA) buffer and loaded onto precast 12% sodium dodecyl sulfate polyacrylamide gels. After separation, the proteins were transferred to nitrocellulose membranes and blocked for 1 h in 5% skim milk diluted in Tris-buffered saline with 0.1% Tween 20. The membranes were incubated overnight with the following primary antibodies: mouse anti-β-actin (ab3280, 1 : 2000; Abcam, Cambridge, U.K.), rabbit anti-AQP-5 (AB15858, 1 : 2000; Millipore, Merck), rabbit anti-caspase-3 (9661S, 1 : 2000; Cell Signaling Technology, Danvers, MA, U.S.A.), mouse anti-Bax (sc-7480, 1 : 2000; Santa Cruz, Dallas, TX, U.S.A.), and rabbit anti-Bcl-2 (sc-492, 1 : 2000; Santa Cruz). Next, the membranes were washed 3 times with Tris-buffered saline with Tween 20. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (7074S, 1 : 2000) or anti-mouse IgG (7076S, 1 : 2000) antibody from Cell Signaling Technology. Protein bands were visualized using ECL Plus substrate (Thermo Fisher Scientific), and band densities were measured using Quantity One software (PDI, New York, NY, U.S.A.).
Evaluation of Mitochondrial Membrane PotentialThe mitochondrial membrane potential was assessed using the JC-1 MitoMP detection kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, A253 cells were cultured in black/clear-bottom 96-well plates (Corning Incorporated, Corning, NY, U.S.A.) at a density of 1.5 × 104 cells/100 µL/well using serum-free medium and preincubated with or without 1 or 10 µg/mL of GB sample solution for 2 h before TNF-α exposure. At the end of the experiment, 100 µL of JC-1 working solution was added to each well and the plate was incubated at 37 °C for 30 min. After incubation, the cultures were washed twice with Hank’s balanced salt solution, followed by the addition of 100 µL of imaging buffer solution. The fluorescence intensity of the cultures was measured using a fluorescence microplate reader (Infinite M200, TECAN, Männedorf, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (green fluorescence) and at an excitation wavelength of 535 nm and an emission wavelength of 595 nm (red fluorescence). The ratio of the red and green fluorescence intensities was calculated to determine mitochondrial membrane potential.
Caspase-3 Activity AssayCaspase-3 activity was measured using Amplite™ Fluorimetric caspase 3/7 assay kit (AAT Bioquest, Sunnyvale, CA, U.S.A.) according to the manufacturer’s protocol. Briefly, A253 cells were seeded in black/clear-bottom 96-well plates at a density of 1.5 × 104 cells/100 µL/well and incubated for 24 h. After incubation with or without GB sample solution for 2 h, the medium was completely changed, and the cultures were treated with TNF-α for 24 h. Following the addition of the caspase 3/7 assay solution, the cultures were incubated for 1 h at room temperature protected from light. Increase in fluorescence increase at an excitation wavelength of 490 nm and an emission wavelength of 525 nm was monitored using a fluorescence microplate reader.
Determination of ROS LevelsIntracellular ROS levels were determined using the Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Briefly, A253 cells were seeded in black/clear-bottom 96-well plates at a density of 1.5 × 104 cells/100 µL/well and preincubated with or without GB sample solution (1 or 10 µg/mL) in serum-free medium for 2 h. TNF-α was then added to the medium, and the cultures were incubated for 24 h. After washing twice with Hank’s balanced salt solution, the cultures were stained with 10 µM of DCFH-DA at 37 °C for 30 min protected from light. Fluorescence intensity was measured at an excitation wavelength of 490 nm and an emission wavelength of 535 nm using a fluorescence microplate reader.
Histone Acetylation AssayHistones from A253 cells were extracted using an acid extraction method following the manufacturer’s protocol for acetylated H3 and H4 histones (Epigentek Group, Farmingdale, NY, U.S.A.). The extracted histones were then incubated with specific antibodies. The levels of acetylated histone H4 were subsequently determined using the EpiQuik™ total histone acetylation detection assay kit (Epigentek Group). Blank samples did not include nuclear extracts. Fluorescence intensity was measured at an excitation wavelength of 520 nm and an emission wavelength of 590 nm using a fluorescence microplate reader. The H4 acetylation level was calculated as follows: acetylation = relative fluorescence units (treated sample − blank)/relative fluorescence units (untreated sample − blank).
Statistical AnalysisData were presented as means ± standard deviation of 3 independent experiments and analyzed using JMP Pro software 15.0.0 (SAS Institute, Cary, NC, U.S.A.). Statistical significance was assessed using Dunnett’s test and Student’s t-test, with a p < 0.05 considered statistically significant.
This study initially conducted a cytotoxicity assay of GBJ on A253 cells. We treated the cells with different GBJ concentrations (range: 1–100 µg/mL) for 24 h and then assessed them using CCK-8 assay. At a GBJ concentration of 1 µg/mL, the cell survival rate only slightly decreased to 95.82 ± 2.53%, and the rate remained at 88.66 ± 3.37% at 10 µg/mL. However, the cell survival rate notably dropped, reaching its lowest value of 53.70 ± 4.51%, when the concentration increased to 50 or 100 µg/mL.
GBJ Pretreatment Prevents TNF-α-Mediated Reduction in AQP-5 mRNA Expression in A253 CellsWe first measured the AQP-5 mRNA levels in A253 cells using RT-PCR. Quantification of the band intensities of RT-PCR products revealed that TNF-α treatment reduced the AQP-5 mRNA level to 0.14 ± 0.01 of the control cultures (Fig. 1). In contrast, the AQP-5 mRNA level was higher in cultures pretreated with 10 µg/mL GB sample solution compared to those pretreated with 1 µg/mL GB sample solution, with a value of 0.70 ± 0.06. In contrast, the AQP-5 mRNA level in cultures treated only with 10 µg/mL GB sample solution in the absence of TNF-α was comparable to that in the control cultures. As expected, the AQP-5 mRNA level was comparable between the cultures treated with CR sample solution and those treated with TNF-α.
A253 cells were pretreated with indicated concentrations of goji berry juice (GBJ) or Coptidis rhizome (CR) for 2 h, followed by treatment with tumor necrosis factor alpha (TNF-α) for 24 h. Relative mRNA expression levels of AQP-5 were evaluated by Quantity One software and normalized to GAPDH. Data are presented as means ± standard deviation (n = 3). Groups 3, 4, and 6 were compared using Dunnett’s test. ** p < 0.01 compared to Group 2. Group 5 was compared to Group 1 using Student’s t-test.
By Western blotting, AQP-5 protein level was decreased to 0.20 ± 0.03 in TNF-α-treated cultures compared to the control cultures (Fig. 2). However, pretreatment with 10 µg/mL GB sample solution led to an increase in AQP-5 protein level to 0.78 ± 0.07. The cultures treated only with GB sample solution in the absence of TNF-α exhibited minimal changes in AQP-5 protein level compared to the control cultures. Of note, the AQP-5 protein level in cultures treated with CR sample solution was similar to that detected in the TNF-α-treated cultures.
A253 cells were pretreated with indicated concentrations of GBJ or CR for 2 h, followed by treatment with TNF-α for 24 h. The membrane fraction of cells was isolated, and AQP-5 protein levels were determined using Western blotting and quantified using the Quantity One software. Data are presented as means ± standard deviation (n = 3). Groups 3, 4, and 6 were compared using Dunnett’s test. ** p < 0.01 compared to Group 2. Group 5 was compared to Group 1 using Student’s t-test.
As shown in Fig. 3A, the mitochondrial membrane potential of the TNF-α-treated cultures was decreased in the ratio to 0.80 ± 0.13 compared to the control cultures. However, pretreatment with 10 µg/mL GB sample solution limited the decrease in mitochondrial membrane potential to 2.47 ± 0.48, which was significantly lower compared to the TNF-α-treated cultures. Furthermore, pretreatment with 10 µg/mL GB sample solution inhibited the increase in caspase-3 activity detected in the TNF-α-treated cultures (Fig. 3B). As expected, CR sample solution did not provide protection against the decline in mitochondrial potential or caspase-3 activation.
A253 cells were pretreated with indicated concentrations of GBJ or CR for 2 h, followed by treatment with TNF-α for 24 h. The relative fluorescence intensity of both mitochondrial membrane potential (A) and activity of caspase-3 (B) were determined. Data are presented as means ± standard deviation (n = 3). Groups 3, 4, and 6 were compared using Dunnett’s test. * p < 0.05 and ** p < 0.01 compared to Group 2. Group 5 was compared to Group 1 using Student’s t-test.
By Western blotting, TNF-α treatment for 24 h led to an increase in the proapoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2, indicating the induction of apoptosis. However, pretreatment with 1 or 10 µg/mL GB sample solution before TNF-α treatment led to a reduction in Bax protein level and an increase in Bcl-2 protein level (Fig. 4A). As shown in Fig. 4B, the Bax/Bcl-2 ratio was 11.21 ± 0.09 in the cultures treated with TNF-α and 7.88 ± 0.40 in those pretreated with 10 µg/mL GB sample solution before the TNF-α treatment. Furthermore, the caspase-3 protein level, which increased from 0.15 ± 0.01 in the control cultures to 6.60 ± 0.24 in the TNF-α-treated cultures, was decreased to 3.00 ± 0.35 in the cultures pretreated with GB sample solution (Figs. 4C, D).
A253 cells were pretreated with indicated concentrations of GBJ or CR for 2 h, followed by treatment with TNF-α for 24 h. The protein levels of Bax and Bcl-2 (A) as well as caspase-3 (C) were determined using Western blotting. The relative protein expression levels of Bax and Bcl-2 (B) as well as caspase-3 (D) were evaluated using the Quantity One software. Data are presented as means ± standard deviation (n = 3). Groups 3, 4, and 6 were compared using Dunnett’s test. * p < 0.05 and ** p < 0.01 compared to Group 2. Group 5 was compared to Group 1 using Student’s t-test.
We investigated the effect of GBJ on TNF-α-induced intracellular ROS levels using DCFH-DA fluorescence. As shown in Fig. 5, the intracellular ROS levels were significantly higher in the TNF-α-treated cultures, reaching 8.27 ± 0.29 compared to the control cultures in which the intracellular ROS level was normalized to 1. However, the increase in intracellular ROS levels was limited to 5.75 ± 0.33 in the cultures pretreated with 10 µg/mL GB sample solution before TNF-α treatment.
A253 cells were pretreated with indicated concentrations of GBJ or CR for 2 h, followed by treatment with TNF-α for 24 h. The relative fluorescence intensity of DCFH-DA was determined to assess intracellular reactive oxygen species (ROS). Data are presented as means ± standard deviation (n = 3). Groups 3, 4, and 6 were compared using Dunnett’s test. * p < 0.05 and ** p < 0.01 compared to Group 2. Group 5 was compared to Group 1 using Student’s t-test.
Finally, we examined the extent of histone acetylation. As shown in Fig. 6, the acetylated histone H4 level was decreased to 0.30 ± 0.04 in the TNF-α-treated cultures compared to the control cultures. However, pretreatment with 10 µg/mL GB sample solution partially limited the reduction in acetylated histone H4 level to 0.76 ± 0.08. Moreover, the acetylated histone H4 level was comparable between the cultures treated only with GB sample solution and the control cultures.
A253 cells were pretreated with indicated concentrations of GBJ or CR for 2 h, followed by treatment with TNF-α for 24 h. The levels of acetylated histone H4 were determined using the EpiQuik™ global histone acetylation assay. Data are presented as means ± standard deviation (n = 3). Groups 3, 4, and 6 were compared using Dunnett’s test. ** p < 0.01 compared to Group 2. Group 5 was compared to Group 1 using Student’s t-test.
In the present study, in vitro exposure of TNF-α to human salivary gland cells led to a decrease in both the mRNA and protein expression of AQP-5, which is primarily involved in water transport associated with exocrine secretion in salivary glands. Pretreatment of cells with GBJ suppressed the TNF-α-mediated decrease in AQP-5 expression at both mRNA and protein levels. Additionally, treatment with GBJ alone did not result in a decrease in AQP-5 expression at both mRNA and protein levels, indicating that GBJ by itself might not have a significant inhibitory effect on AQP-5 expression. The expression levels of both AQP-5 mRNA and protein remained low in cells pretreated with Copidis Rhizome. The expression of AQP-5 mRNA is regulated by histone H4 acetylation, which functions as a regulator of chromatin structure and plays a role in transcriptional activation and repression. TNF-α-induced deacetylation of histone H4 has been shown to be associated with decreased AQP-5 mRNA expression.13,17) In the present study, we demonstrated that pretreatment of human salivary gland cells with GBJ effectively prevented the TNF-α-induced deacetylation of histone H4. Overall, these findings suggest the modulation of histone acetylation as a mechanism by which GBJ exerts its influence on AQP-5 expression. However, treating cells with Copidis Rhizome did not significantly affect the acetylation of histone H4 in salivary gland cells.
We also found that TNF-α treatment led to a decrease in mitochondrial membrane potential in human salivary gland cells. TNF-α-mediated reduction in mitochondrial membrane potential has been shown to trigger mitochondria-dependent apoptosis.18,19) In the present study, GBJ pretreatment before TNF-α exposure of the cultures effectively mitigated the reduction in mitochondrial membrane potential, suggesting that GBJ could delay TNF-α-induced apoptosis by preserving mitochondrial membrane potential. Relatedly, apoptosis is regulated by the balance between Bax and Bcl-2, where Bcl-2 inhibits apoptosis19) and plays a role in controlling the mitochondrial cell death signaling pathway activated by Bax.20) We found that GBJ pretreatment led to a decrease in Bax protein levels and an increase in Bcl-2 protein levels, resulting in a reduction of the Bax/Bcl-2 ratio in human salivary gland cells, which suggested that GBJ also inhibited Bax-mediated mitochondria-dependent apoptosis. We also assessed the downstream apoptotic signaling pathway, specifically caspase-3, which plays a pivotal role in the execution of apoptosis. We found that TNF-α treatment resulted in elevated intracellular protein levels and activity of caspase-3, which were attenuated by GBJ pretreatment. Similarly, the evaluation of Copidis Rhizome demonstrated quite similar results to those of cells treated with TNF-α, particularly in association with mitochondrial membrane potential, the Bax/Bcl-2 ratio, intracellular protein levels, and caspase-3 activity in human salivary gland cells. The results indicate that GBJ exhibits a potential to alleviate apoptosis in cells expressing AQP-5 by modulating mitochondrial membrane potential, the Bax/Bcl-2 ratio, and caspase-3.
TNF-α induces the generation of intracellular ROS,21–23) which has also been considered as a mechanism of decreased AQP-5 expression in xerostomia.12) The results revealed that GBJ effectively suppressed TNF-α-induced intracellular ROS generation when compared to Copidis Rhizome.
Altogether, our findings suggested that GBJ exhibited a protective effect in human salivary gland cells in an in vitro model of SS-associated dry mouth. This effect was mediated through the antiapoptotic and antioxidant properties of GBJ. Additionally, GBJ effectively inhibited the decrease in AQP-5 mRNA and protein expression levels, which are related to saliva secretion, and limited the reduction in acetylated histone H4 level. These results highlight a mechanism for the potential protective role of GBJ in oral dryness associated with SS. Our examination of GBJ’s components revealed a significant presence of neoxanthin. However, a comprehensive understanding of how neoxanthin impacts oral dryness in SS remains lacking. Similarly, the effects of other components on oral dryness in this context remain unclear. Considering studies that highlight the antioxidant properties of zeaxanthin and β-cryptoxanthin palmitate,24,25) these compounds show potential, but more research is needed for a clearer understanding. Notably, our experiments were conducted in two dimensional (2D) cultures, and whether the same effects can be observed in salivary gland tissue remains uncertain. Therefore, future studies should determine the potential protective effect of GBJ in 3D salivary gland organoids. Moreover, while there have been reports discussing interleukin-2 (IL-2)-induced inflammation in SS,26) based on the results of our experiments, it’s reasonable to consider that inflammation primarily driven by TNF-α might also be involved. Therefore, we will continue to investigate the preventive effects of GBJ against TNF-α.
This work was supported by JSPS KAKENHI Grant Number: 22K06684.
Masatoshi Takakura received the goji berry juice (100% fruit juice) used in the present study from Iskra Industry Co., Ltd.; Takuya Okamoto and Tong Xuan Fu are employees of Iskra Industry Co., Ltd.
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