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Protective Effects of Cuscutae Semen Extract from Oxidative Stress and Amyloid β25-35-Induced SH-SY5Y Neuronal Cell Dysfunction
J Korean Med Obes Res 2024;24:111-20
Published online December 31, 2024;  https://doi.org/10.15429/jkomor.2024.24.2.111
Copyright © 2024 The Society of Korean Medicine for Obesity Research.

Hyo Jeong Seo1†, Ji-Hyun Kim1†, Yu Ri Kwon1, Yung-Hyun Choi2, Eun Ju Cho1,3, Jeong Sook Noh4

1Department of Food Science and Nutrition, Pusan National University, 2Department of Biochemistry, Dong-eui University College of Korean Medicine, 3BK21 FOUR Program: Precision Nutrition Program for Future Global Leaders, Pusan National University, 4Department of Food Science and Nutrition, Tongmyong University
Eun Ju Cho
Department of Food Science and Nutrition, Pusan National University, 2 Busandaehakro 63beon-gil, Geumjeong-gu, Busan 46241, Korea
Tel:+82-51-510-2837
Fax: +82-51-583-3648
E-mail: ejcho@pusan.ac.kr

Jeong Sook Noh
Department of Food Science and Nutrition, Tongmyong University, 428 Sinseon-ro, Nam-gu, Busan 48520, Korea
Tel: +82-51-629-1716
Fax: +82-51-629-2229
E-mail: jsnoh2013@tu.ac.kr

†The authors contributed equally to this work as co-first authors.
Received August 2, 2024; Revised October 25, 2024; Accepted November 1, 2024.
cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Objectives: Amyloid beta (Aβ) is generated by the amyloidogenic pathway. Its accumulation is a major cause of Alzheimer’s disease. The present study investigates the free radical scavenging activities and inhibition of Aβ25-35-induced neurotoxicity by Cuscutae semen (CS) water extract treatment.
Methods: The scavenging activities of radicals, including 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), and nitric oxide (NO) of the CS water extract were assessed. Moreover, the SH-SY5Y cells were used to investigate cell viability, inflammation-related protein expression, and inflammatory cytokine secretion using CS water extract to determine neuroprotective effects.
Results: The CS water extract inhibited the production of free radicals such as DPPH, OH, and NO, dose- dependently. Meanwhile, oxidative stress was induced, and cell viability was decreased by Aβ25-35. The CS water extract treatment significantly increased cell viability in Aβ25-35-stimulated SH-SY5Y cells. Additionally, the treatment with CS water extract significantly down-regulated the expression of inducible nitric oxide synthase and cyclooxygenase-2 in Aβ25-35‐stimulated SH-SY5Y cells. Secretion of interleukin-1β and interleukin-6 in Aβ25-35‐induced SH-SY5Y cells was significantly inhibited by CS water extract.
Conclusions: This study indicated the free radical scavenging activities and neuroprotective effects of CS water extract.
Keywords : Alzheimer disease, Beta amyloid, Antioxidants, Free radicals, Inflammation, Oxidative stress
Introduction

Oxidative stress occurs due to an imbalance between pro-oxidants and antioxidants, resulting from an uneven removal and production of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Various antioxidant systems play endogenous roles in the body, helping to protect against free radical damage by scavenging excess ROS and RNS1). However, if this antioxidant system is broken, the ROS and RNS are overproduced, leading to oxidative damage to deoxyribonucleic acid, proteins, and lipids2). Additionally, oxidative stress—a crucial factor in neuronal cell death—induces a variety of diseases, such as inflammatory illnesses and diverse degenerative diseases related to aging3-5).

The amyloid beta (Aβ) precursor protein (APP) stimulates the production of Aβ, which results in oxidative injury to neuronal cells6). This oxidative stress increases inflammationelated protein expressions such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), eventually triggering cell death. Aβ peptide initiates inflammation in the hippocampus of the brain, which is a major cause of Alzheimer’s disease (AD). In the advancement of AD, build-up and aggregation of Aβ peptide in the hippocampus generally results in the activation of glial cells. This, in turn, begins a neuroinflammatory response including reactive oxygen intermediates and inflammatory cytokines involving interleukin-1β (IL-1 β), IL-6, and tumor necrosis factor7). Therefore, Aβ directly causes the pathogenesis of AD. Meanwhile, SH-SY5Y is a human neuronal cell line commonly used in scientific studies such as in vitro models of neuronal differentiation and function. Studies have confirmed that oxidative stress is involved in cell death and inflammation using SH-SY5Y neuronal cells8). Therefore, Aβ fragment 25-35 (Aβ25-35)-treated SH-SY5Y cells are being used to study materials with protective effects under oxidative stress.

The seed of Cuscuta chinensis L am. (Cuscutae semen, Convolvulaceae; CS) is a medicinal herb with remarkable pharmacological activities, including antioxidant9), anti-inflammatory10), anti-obesity11), anti-neurotoxicity12), and antiapoptotic activities13). Many studies on CS have explored its medicinal features and mechanisms. In previous studies, the protective effects of flavonoids from CS on oxidative stress were investigated to understand the underlying mechanisms. These studies revealed that CS treatment has the ability to scavenge ROS in a tube test, and enhances the activity of antioxidant enzymes in PC12 cells from damage condition induced by H2O2 14). Furthermore, astragalin is a major flavonoid from CS, with implications in various pharmacological activities. It has been reported that astragalin from CS significantly increases antioxidant enzyme activities and inhibits inflammation15).

However, studies on the protective effects of CS treatment against neuronal injury induced by Aβ25-35 remain scarce. Therefore, the present study confirmed the antioxidative effects of the CS water extract in an in vitro system such as the 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), and nitric oxide (NO) radical scavenging abilities. Additionally, the protective mechanisms of CS water extract associated with inflammation were investigated in Aβ25-35-treated SH-SY5Y neuronal cells.

Materials and Methods

1. Reagent

Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Aβ25-35, DPPH, and 2-deoxy-ribose purchased from Sigma-Aldrich (St. Louis, MO, USA). H2O2 was bought from Junsei (Tokyo, Japan). FeSO4⋅7H2O was obtained from the Daejung Chemicals & Metals Co. Ltd (Siheung, Korea). EDTA disodium salt dehydrates, and phosphoric acid were purchased from Samchun Pure Chemical Co. Ltd (Pyeongtaek, Korea). Sodium pentacyanonitrosylferrate (III) dihydrate (SNP) was bought from the Junsei. Trichloroacetic acid (TCA) was purchased from Biosesang (Seoul, Korea). Thiobarbituric acid (TBA) was obtained from Acros Organics (Morris Plains, NJ, USA). Phenazine methosulfate and NADH were purchased from Bio Basic (New York, USA). Radioimmunoprecipitation assay (RIPA) buffer was purchased from Elpis Biotech (Daejeon, Korea). A protease inhibitor cocktail was supplied by Calbiochem (Cambridge, MA, USA). Polyvinylidene fluoride (PVDF) membranes were procured from Millipore (Billerica, USA). The primary antibodies such as iNOS and COX-2 were purchased from Santa Cruz (MA, USA). The secondary antibody was purchased from Cell Signaling Biotechnology (Beverly, MA, USA). Enhanced chemiluminescence (ECL) substrate solution was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Human IL-1β/IL-1F2 DuoSet ELISA, Human IL-6 DuoSet ELISA were bought from R&D systems (Minneapolis, MN, USA).

2. Preparation of CS water extract

The CS water extract was obtained from an agricultural company corporation, Herbal company (Gyeonggi-do, Korea). The dried CS was added to 20 times their weight in water and extracted using hot water for 2 hours. The extract was then concentrated by evaporation at 40 °C and freeze-dried to produce samples. Afterward, the extract was dissolved in DMSO and used in the experiment.

3. DPPH radical scavenging assay

The DPPH radical scavenging activity of CS water extract was assessed following the method of Hatano et al.16). Each concentration of the sample (10, 25, 50, and 100 μM) was added to a 60 μM DPPH solution in a 1:1 ratio in 96-well plates. After blocking the light in a darkroom, the mixture was incubated at room temperature for 30 minutes. The absorbance was then measured at 540 nm using a microplate reader (Thermo Fisher Scientific, Vantaa, Finland).

4. OH radical scavenging assay

The OH radical scavenging activity was measured according to the method reported by Gutteridge2). Each different concentration of CS water extract (10, 25, 50, and 100 μM) was added to the reaction mixture containing 10 mM FeSO4⋅H2O-EDTA with 10 mM 2-deoxyribose solution and 10 mM H2O2, and thereafter, incubated at 37 °C for 4 h. After incubation, 2.8% TCA and 1.0% TBA solutions were added to the mixture. After boiling at 100 °C for 20 min, ice-cooling was employed for 10 min. The OH radical scavenging activity was measured using an RT-6100 microplate reader at 490 nm (Rayto Life and Analytical Sciences Co. Ltd., Shenzhen, China).

5. NO radical scavenging assay

Sodium nitroprusside (5 mM) in phosphate-buffered saline (PBS, pH 7.4) was mixed with different concentrations of CS water extract (10, 25, 50, and 100 μM) dissolved in MeOH, and mixed with SNP (10 mM) and incubated at room temperature exposed to light for 150 min. The samples from the above were reacted with Griess reagent (1% sulfanilamide, 2% H3PO4, and 0.1% naphthyl ethylenediamine dihydrochloride) as 100 μl each added at a ratio of 1:1 in a 96-well plate. It reacted for 10 min in a dark environment. After 10 min, the absorbance of the chromophore formed during the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthyl ethylenediamine dihydrochloride was read at 540 nm using an RT-6100 microplate reader (Rayto Life and Analytical Sciences Co. Ltd.). It was indicated that the absorbance of standard solutions of potassium nitrite was treated in the same manner with Griess reagent.

6. Cell culture

The human-derived SH-SY5Y neuron cell (Korean cell line bank, Seoul, Korea) used a dulbeccós modified eagle medium medium containing 100 units/mL of penicillin streptomycin and 10% fetal bovine serum at T-75-flask, and was cultured at 37 ℃, 5% CO2 incubator. With cells in a more than 80% differentiated state, after cleaning the cells using PBS (pH 7.4), and then 0.05% trypsin containing 0.02% EDTA, the attached cells were separated. The cells were centrifuged at 1000 rpm for 3 min to collect cells and were evenly distributed in the media and used in the experiment.

7. Cell viability

The cell survival rate progressed according to the MTT method17). When cells are more than 80% confluence, after seeding to 96-well plates at a density of 2.5 × 105 cells/mL and incubating 5% CO2 at 37 ºC for 24 h. The cells were treated with CS water extract at three concentrations (10, 50, and 100 μg/mL), and incubated for 2 h. Next, Aβ25-35 at the concentration of 25 μM was added to the cells. After reaction for 24 h, 200 μL of MTT solution (concentration of 5 mg/mL) w as a dded to t he c ells. After 4 h o f incubation, the MTT solution (reaction solution) was removed, and DMSO was added. Formazan crystals were dissolved in 200 μL of DMSO for 30 min at room temperature. After solubilization, the absorbance of the formazan was measured at 540 nm using a microplate absorbance reader (Thermo Fisher Scientific).

8. Western blotting analysis

Seeding the cell was played at 1 × 106 cells/mL in a cell culture dish (90 × 20 mm). It was cultured for 24 h, and thereafter, treated with CS water extract (10, 50, and 100 μ g/mL). After incubation for 2 h, it was treated at a concentration of Aβ25-35 (25 μM). After 24 h, the cells were collected, and RIPA buffer with protease inhibitor cocktail was added, centrifuged at 4 °C, 12,000 rpm, 30 min, and the supernatant was transferred to the e-tube. Next, samples used for analysis were prepared by mixing the cell supernatant containing an equal amount of protein and the sample buffer (1:1, v/v). The sample underwent electrophoresis for 2 h and 90 V at 10~13% sodium dodecyl sulfate polyacrylamide gels and transferred to the PVDF membrane for 2 h. The membrane was blocked for 1 h with 5% skim milk, washed for 10 min and three-times with PBS-tween-20, and reacted overnight at 4 °C with the primary antibody (iNOS, 1:200, COX-2, 1:500 concentration). Thereafter, the secondary antibody was reacted at room temperature for 1 h, and the ECL solution was added to measure protein expression using the Davincichemiluminescent imaging system (CoreBio, Seoul, Korea).

9. Cytokine assays

The cells were confluenced by more than 80%, seeding at 2.5 × 105 cells/mL on 96-well plates, and then incubated for 24 h before they were treated with CS water extract (10, 50, and 100 μg/mL). After incubation for 2 h, they were treated at a concentration of Aβ25-35 (25 μM). After incubating the treated samples for 24 h, an experiment was conducted using the medium supernatant according to the kit method.

10. Statistical analysis

Data in this study are presented as mean±standard deviation and analyzed using the SPSS 26.0 software (IBM Corporation, NY, USA). One-way analysis of variance was employed for statistical processing of the data. P<0.05 suggested that the difference was statistically significant.

Results

1. DPPH radical scavenging activity

CS water extract confirmed a dose-dependently increased DPPH radical scavenging activity (Table 1). Particularly, CS water extract showed a DPPH radical scavenging activity of 79.96% at doses of 100 μg/mL. These results indicated that CS water extract has the DPPH radical scavenging activity.

DPPH Radical Scavenging Activity of CS Water Extract

Scavenging activity (%)
Treatment (μg/mL)CS water extract
1022.18±3.00d
2545.97±3.03c
5070.20±4.21b
10079.96±2.68a

The results are presented as mean±standard deviation.

DPPH: 2,2-diphenyl-1-picrylhydrazyl, CS: Cuscutae semen.

The different letters (a, b, c, d) are significantly different (P<0.05) by Duncan’s multiple range test.

2. OH radical scavenging activity

OH radical is the most reactive of the ROS. It causes severe injuries to adjacent biomolecules, leading to oxidative damage to DNA, lipids, and proteins2). The results of the OH radical scavenging activity of CS water extract are indicated in Table 2. The CS water extract showed dose-dependently elevated OH radical scavenging activity in 41.40%, 66.92%, 80.93%, and 89.78%, respectively (10, 25, 50, and 100 μg/mL). Particularly, the scavenging activity at 50 μ g/mL and 100 μg/mL were more than 80%.

OH Radical Scavenging Activity of CS Water Extract

OH radical scavenging activity (%)
Treatment (μg/mL)CS water extract
1041.40±0.43d
2566.92±0.50c
5080.93±0.43b
10089.78±3.11a

The results are presented as mean±standard deviation

OH: hydroxyl, CS: Cuscutae semen.

The different letters (a, b, c, d) are significantly different (P<0.05) by Duncan’s multiple range test.

3. NO radical scavenging activity

The investigated results of NO radical scavenging activity of CS water extract are indicated in Table 3. The percentage inhibition of NO radicals by the CS water extract effects concentration-dependently increased NO radical scavenging activity by 35.25%, 37.12%, 41.10%, and 42.84%, respectively (10, 25, 50, and 100 μg/mL), with the highest inhibition of 41. 10% at 1 00 μ g/mL. T hese r esults s how that C S water extract has antioxidant capabilities for scavenging NO radicals.

NO Radical Scavenging Activity of CS water extract

NO radical scavenging activity (%)
Treatment (μg/mL)CS water extract
1035.25±0.46d
2537.12±0.62c
5041.10±0.62b
10042.84±0.39a

The results are presented as mean±standard deviation.

NO: nitric oxide, CS: Cuscutae semen.

The different letters (a, b, c, d) are significantly different (P<0.05) by Duncan’s multiple range test.

4. Effect on cell viability in Aβ25-35 treated SH-SY5Y cells

After treating Aβ25-35 of 25 μM in SH-SY5Y neuron cells, cell viability was measured. It was confirmed that the cell viability of the control group (79.26%) treated with Aβ 25-35 was significantly reduced compared to that of the normal group (100%), which was Aβ25-35 non-treated, leading to damage to cell viability by Aβ25-35 (Fig. 1). However, the cell viability significantly increased at all concentrations when treated with CS water extract. Particularly, the cell viability was 90.20% and 90.41%, respectively, at CS water extract 50 μg/mL and 100 μg/mL treatments. It confirmed the effect of protecting nerve cells by improving the damaged cell viability via CS water extract treatment.

Fig. 1. Effect of CS water extract on cell viability in Aβ25-35-treated SH-SY5Y cells. The results are presented as mean±standard derviation. The different letters (a, b, c) are significantly different (P<0.05) by Duncan’s multiple range test. CS: Cuscutae semen, Aβ25-35: amyloid beta fragment 25-35.

5. Effect of CS water extract on iNOS and COX-2 expression in Aβ25-35 treated SH-SY5Y cells

To confirm the anti-inflammatory effect of the CS water extract, Aβ25-35 was treated on SH-SY5Y neurons, and thereafter, the expressions of inflammation-related proteins iNOS (Fig. 2A) and COX-2 (Fig. 2B) were measured. The experiment confirmed that the control group significantly increased the expression of iNOS and COX-2 proteins compared to the normal group, indicating that an inflammatory response was induced owing to Aβ25-35. Conversely, in the group treated with CS water extract, iNOS and COX-2 expressions were significantly decreased. Particularly, the expression of COX-2 in the 100 μg/mL CS water extract treatment group was decreased to a level similar to that of the normal group, thereby confirming the effect of suppressing inflammation of CS water extract.

Fig. 2. Effect of CS water extract on iNOS and COX-2 expression in Aβ25-35-treated SH-SY5Y cells. (A) iNOS protein expression, (B) COX-2 protein expression. The results are presented as mean±standard derviation. The different letters (a, b, c, d) are significantly different (P<0.05) by Duncan’s multiple range test. CS: Cuscutae semen, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, Aβ25-35: amyloid beta fragment 25-35.

6. Effect of CS water extract on IL-1β and IL-6 in Aβ25-35 treated SH-SY5Y cells

To investigate the anti-inflammatory effect of the CS water extract, Aβ25-35 was treated on SH-SY5Y neurons, and thereafter, the concentrations of inflammation-related cytokines IL-1β (Fig. 3A) and IL-6 (Fig. 3B) were measured. The control group significantly increased the concentrations of IL-1β and IL-6 compared to the normal group, leading to an inflammatory response. However, the concentrations of IL-1β and IL-6 were significantly reduced at all groups treated with CS water extract. Particularly, at 100 μg/mL, the released cytokine concentration was 10.47 pg/mL in IL-1β and 13.87 pg/mL in IL-6, confirming the effect of inhibiting inflammation of the CS water extract.

Fig. 3. Effect of CS water extract on IL-1β and IL-6 in Aβ25-35-treated SH-SY5Y cells. (A) IL-1β, (B) IL-6. The results are presented as mean±standard derviation. The different letters (a, b, c) are significantly different (P<0.05) by Duncan’s multiple range test. CS: Cuscutae semen, IL-1β: interleukin-1beta, IL-6: interleukin-6, Aβ25-35: amyloid beta fragment 25-35.
Discussion

Obesity strongly increases the risk of acute and chronic disease, such as hypertension, diabetes, and insulin resistance, and approximately 2.8 million individuals die annually because of obesity-related complications18). Particularly, o besity i nduces the cerebral oxidative stress, which contributes substantially to the neurodegeneration, including AD19). Andrade et al.20) reported that obesity is associated with morphological changes in the brain, including in the hippocampus and frontal cortex. Therefore, the neuroprotective effect of medicinal herbal substances is promising therapy for people with obesity-driven comorbidities that can alleviate neurological disorder.

Meanwhile, as mentioned before, diverse studies have reported that CS extracts using the kinds of solvents have been investigated their physiological and pharmacological activities. In our preliminary study, we compared the SH-SY5Y cell viability by treatment of CS-water extract and CS-ethanol extract in Aβ25-35-induced neurotoxicity. The result indicated that the CS-water extract showed slightly higher neuroprotective effect than CS-ethanol extract. For this reason, in present study, we demonstrated whether CS water extract could have a protective effect against oxidative stress and Aβ-induced neurotoxicity.

Inflammation contributes to the pathophysiological mechanism of AD21) and various environmental elements, such as antioxidants and proteoglycans, modify Aβ toxicity. Aβ is related to ROS generation, which causes mitochondrial dysfunction, lipid peroxidation, and inflammation22). Therefore, removal of excess ROS or inhibition of their generation by antioxidants is an effective method of preventing oxidative cell death. Recently, it was confirmed that antioxidant supplementation can interfere with the progression of neurodegenerative diseases and alleviate nerve damage caused by free radicals23). Particularly, natural antioxidants derived from various plants are receiving increasing attention for their potential in the prevention and treatment of neurodegenerative diseases.

The DPPH radical scavenging activity is a method to analyze the antioxidant capacity of biological substrates24). This assay was performed by dropping an antioxidant and samples onto a 96-well plate. DPPH is a stable free radical at room temperature that accepts an electron/hydrogen radical, converting it into a stable diamagnetic molecule25). Through the antioxidant, the DPPH radicals become stable DPPH molecules, changing in color from deep violet to pale yellow. The violet color intensity of DPPH was inversely proportional to the antioxidant activity of the samples26). The decreased capability of DPPH radicals was measured by reducing absorbance, which is induced by antioxidants. The most reactive free radical, the OH radical, is formed by a Fentontype reaction, and causes severe damage to adjacent biomolecules27). Neuroinflammatory processes induced by proinflammatory cytokines showed increased ROS and RNS, in addition to other unknown components that have neurotoxic properties. Neurotoxic amounts of RNS are formed by the activity of iNOS28). The produced iNOS catalyzes the formation and release of a significant amount of NO, which then plays a key role in disease pathophysiology29).

In our experimental results, significant scavenging activity was observed for CS water extract. The CS water extract showed DPPH, OH, and NO radicals scavenging activities. The DPPH, OH, and NO radicals scavenging activity of CS water extract had dose-dependently increased, and especially the OH radical scavenging activity of CS water extract at concentrations of 50 μg/mL and 100 μg/mL was higher than 80%. These results supported that CS water extract had antioxidant capabilities of DPPH, OH, and NO radicals scavenging and could be an antioxidant for protecting from oxidative stress.

SH-SY5Y cells are a kind of human neuronal cell and have been used for the study of neurodegenerative damage in cellular models for neurotoxicity experiments30). It is utilized to study oxidative stress-induced inflammation and apoptosis and to prove the protective effect of samples under oxidative stress31,32). However, the protective effects of CS against Aβ25-35-induced oxidative stress and neurotoxicity have not been studied to date. In this study, we focused on the protective effects of CS under oxidative stress and Aβ 25-35-induced damage in SH-SY5Y neuronal cells.

The MTT tetrazolium reduction assay is a colorimetric assay for assessing cell metabolic activity. The MTT colorimetric assay utilizes the well-established and widely used MTT reagent to determine viable cell numbers in proliferation and cytotoxicity studies. Zraika et al.33) reported that Aβ treatment dose-dependently decreased cell viability in SH-SY5Y cells, indicating that Aβ induces cytotoxicity in neuronal cells. Consistent with this previous study, our results also demonstrated that treatment with Aβ reduced cell viability in SH-SY5Y cells. However, pretreatment with CS water extract effectively restored cell viability, indicating that CS water extract attenuated the cytotoxicity induced by Aβ25-35.

COX-2, activated at the site of inflammation, is rapidly induced by proinflammatory cytokines. It can be broken down into prostaglandin E2 in the body, which plays a key role in inflammation34). Overproduction of inflammatory mediators causes many diseases related to nervous disorders. iNOS catalyzes the reaction of producing NO from L-arginine as a class of enzymes35). Stimuli such as cytokines and lipopolysaccharide result in the expression of iNOS and COX-2 to produce NO and prostaglandin E2 to cause inflammation36). In the study conducted by Nathan et al.37), iNOS was discovered in the brains of mice with AD-like disease resulting from transgenic expression of mutant human APP and PS1. It was a major instigator of Aβ deposition and disease progression. Therefore, inhibition of the production of these inflammatory mediators may suppress diverse inflammatory diseases, including nervous disorders such as AD. In this study, CS water extract inhibited iNOS and COX-2 expression in Aβ25-35-treated SH-SY5Y cells. This result indicated that CS-water extract could be beneficial for preventing the neuroinflammation by Aβ25-35. Kang et al.38) reported that CS extract significantly inhibited the production of prostaglandin E2 by suppressing COX-2 expression in lipopolysaccharide-stimulated BV-2 microglial cells.

The pro-inflammatory cytokines such as IL-1α and IL-1β belong to the IL-1 group of cytokines, which are synthesized as precursor proteins39). Recent studies have shown the increase of IL-1β and IL-6 in AD brains40). Varinthra et al.41) identified that Aβ induced the expression and increased the production of the pro-inflammatory cytokines IL-1β and IL-6. We examined the levels of these proteins quantitatively. The present study indicated that the Aβ25-35-treated SH-SY5Y cells significantly increased the expression of iNOS and COX-2, and the concentration of IL-1β and IL-6 compared with the Aβ25-35-non-treated SH-SY5Y cells. However, treatment with CS water extract significantly decreased it.

Conclusion

In conclusion, CS water extract had antioxidant capabilities of DPPH, OH, and NO radical scavenging. Moreover, CS water extract increased the cell viability in Aβ25-35-treated SH-SY5Y cells, and down-regulated the inflammationrelated protein expression and cytokine concentration, which determined protective effects against Aβ25-35-treated oxidative stress in SH-SY5Y cells. Therefore, this study demonstrated that CS water extract has a protective effect from A β25-35-treated neuronal damage via inhibition of oxidative stress and inflammation.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2020R1I1A207458612).

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

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