Lunasin and total proteins from Glycine soja and Glycine max inhibit inflammation pathways, NF-κB and hippo, in vitro through decreased expression of pro-inflammatory kinases and cytokines

Kusumah, Jennifer

Permalink

https://hdl.handle.net/2142/129894 Copy

Description

Title

Lunasin and total proteins from Glycine soja and Glycine max inhibit inflammation pathways, NF-κB and hippo, in vitro through decreased expression of pro-inflammatory kinases and cytokines

Author(s)

Kusumah, Jennifer

Issue Date

2025-07-18

Director of Research (if dissertation) or Advisor (if thesis)

Gonzalez de Mejia, Elvira

Doctoral Committee Chair(s)

Cadwallader, Keith R

Committee Member(s)

• Chen, Hong
• Madak-Erdogan, Zeynep

Department of Study

Food Science & Human Nutrition

Discipline

Food Science & Human Nutrition

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• soybean
• glycine max
• glycine soja
• inflammation
• NF-kB pathway
• Hippo pathway
• proteins
• peptides
• lunasin
• bioaccessibility
• INFOGEST
• digestion
• in vitro

Abstract

Plant bioactive compounds such as proteins and peptides have received increased attention due to their potential health benefits, including mitigating inflammation. Soybean (Glycine max) is a legume that was first cultivated in China and has since then been widely consumed around the world. The first aim of this research was to evaluate and compare the anti-inflammatory responses of different soybean accessions after simulated gastrointestinal digestion, with special consideration of the glycinin:β-conglycinin ratio. Digests of commercial soybeans (G. max) inhibited the pro-inflammatory enzymes cyclooxygenase (COX)-2 (25–82%), 5-lipoxidase (LOX) (18–35%), and inducible nitric oxide (iNOS) (8–61%); accessions PI245088 glycinin-null (GN) 3, and PI245093 were the most effective inhibitors. Digests of accessions PI245081 and glycinin-null (GN) 1 significantly (p < 0.05) reduced nitrite production by 44 and 47%, respectively, compared to lipopolysaccharide (LPS)-stimulated macrophages. Soybean digests effectively reduced pro-inflammatory cytokine interleukin (IL)-6 (50 and 80% for accessions PI245096 and GN1, respectively). Western blot results showed a decrease in the expression of iNOS, p65, and p50. The glycinin:β-conglycinin ratio was in the range of 0.05–1.57; higher ratio correlated with higher production of IL-1β (r = 0.44) and tumor necrosis factor-alpha (TNF-α, r = 0.56). Inflammatory microarray results showed a significant decrease in the expression of markers granulocyte- macrophage colony-stimulating factor (GM-CSF) and IL-6 in cells treated with soybean digest of accession GN1 compared to LPS. The results suggested that soybean colonic digests exerted their anti-inflammatory potential through nuclear factor kappa B (NF-κΒ) pathway inhibition by decreasing the levels of NF-κB-dependent cytokines and subunits, and inhibition of pro-inflammatory enzyme activity. Higher β-conglycinin content correlated with higher anti-inflammatory potential. The second aim was to compare lunasin and protein concentrations in different accessions of wild-type (G. soja) and commercial soybean G. max, determine the bioaccessibility of lunasin after digestion, and the bioactivity of the peptides produced. Lunasin and protein concentrations showed significant differences among varieties (p < 0.05) and were moderately correlated (r = 0.421). The results showed that the soluble protein concentration of G soja ranged from 166.9 ± 1.5 mg/g to 550.4 ± 32.7 mg/g of defatted flour (DF) with an average of 348.4 ± 77.7 mg/g of DF, while in G. max ranged from 70.0 ± 3.4 mg/g to 842.9 ± 46.9 mg/g of DF with an average of 336.2 ± 153.9 mg/g of DF. Protein profile analysis showed that some accessions had significant differences at a molecular weight around 70 kDa, indicating differences in storage protein β-conglycinin. Lunasin concentration of all G. soja accessions ranged from 8.3 ± 0.1 mg/g to 32.9 ± 1.7 mg/g of DF with an average of 20.9 ± 4.5 mg/g of DF, representing 2 – 13% of total extracted protein, compared to 7.9 ± 0.5 mg/g – 114.4 ± 10.0 mg/g of DF with an average of 34.9 ± 23.3 mg/g of DF in G. max representing an average of 12% of total extracted protein. After digestion, 2.6 – 7.6% of lunasin (average 4.5%) and >50% soluble protein remained intact. Bioactive peptides, sequenced using LC-MS-MS and MassLynx software, were found in the digests: EY (Glu-Tyr), EF (Glu-Phe), ALGLN (Ala-Leu-Gly-Leu-Asn), GAR (gly-Ala-Arg), LTEFL (Leu-Thr-Glu-Phe-Leu), TLEFL (Thr-Leu-Glu-Phe-Leu), and EQAQER (Glu-Gln-Ala-Gln-Glu-Arg). Two of them (EY and TLEFL) were previously found in G. max digests. Peptides were found to be angiotensin-converting enzyme (ACE), renin, and dipeptidyl peptidase IV (DPP-IV) inhibitors and hypolipidemic. G. max differed significantly from G. soja in the bioavailability of protein and lunasin after simulated gastrointestinal digestion, with G. max having lower protein bioavailability but higher lunasin bioavailability. Based on these results, we concluded that different accessions of both commercial and wild-type soybean have different protein profiles, protein and lunasin concentrations, and were able to mitigate inflammation. Analysis of digests revealed that lunasin and soluble protein produced bioactive peptides that inhibit ACE and DPP-IV. This study increased the understanding of lunasin production in soybean ancestral lines, which could be used to develop new commercial accessions with higher lunasin concentration. Lunasin was shown to survive digestion, which is important for its bioaccessibility to exert anti-inflammatory benefits. Our third aim was to elucidate the potential anti-inflammatory mechanisms of enriched wild-type soybean lunasin protein extract (ELPE) in comparison to digested soybean whole flour (DWF), total soluble soybean protein extract (TPE), and lunasin-free soybean protein extract (LFP) on the Hippo pathway, using a human monocytic cell line derived from an acute monocytic leukemia patient (THP-1) as the inflammation model. The results showed that ion-exchange chromatography produced ELPE with lunasin purity that ranged between 56% to 73% and significantly increased lunasin concentration in ELPE accessions PI407207, PI407159, PI407018 and PI424088 (51.6 – 86.9 mg/g of defatted flour, DF) compared to TPE (16.2 – 32.9 mg/g DF). TPE of PI424088 was able to significantly decrease IL-6, MCP-1 and TNF-α production (96%, 76%, 52% respectively). G. soja of all accessions and sample types were most effective at inhibiting IL-6 production (74% - 98%) compared to MCP-1 (6% - 99%). ELPE of PI407207 and TPE of PI424088 significantly (p < 0.05) decreased expression of dephosphorylated YAP1 and increased expression of phosphorylated YAP1 (p < 0.05), as well as significantly (p < 0.05) increasing cytoplasmic YAP1 retention. DWF of PI407207, PI407018, PI407159 significantly (p < 0.05) decreased expression of dephosphorylated LATS1/2 and increased expression of phosphorylated LATS1/2 (p < 0.05). G. soja treatments inhibited inflammation by decreasing pro-inflammatory cytokines IL-6, IL-1β and MCP-1, phosphorylating Hippo core kinases YAP1 and LATS1/2, as well as increasing cytoplasmic YAP1 retention. In conclusion, different accessions of both commercial (G. max) and wild-type (G. soja) soybean have different protein profiles, as well as different protein and lunasin concentrations, and were able to mitigate inflammation through lowering the levels of pro-inflammatory cytokines production, as well as modulating NF-κB and Hippo pathways by decreasing the protein expression of their dephosphorylated core kinases. The results of this research provide more insight into the effect of soybean protein profile after digestion for its anti-inflammatory properties and could be used to guide growers and producers to select the most appropriate soybean cultivar to provide for consumers. This study increased the understanding of lunasin and protein production as well as their bioactivity in soybean ancestral lines, which could be used to develop new commercial varieties with desired characteristics to contribute to new functional food ingredients. Understanding the effect of soybean proteins and peptides on the Hippo pathway, in addition to the classical NF-κB, could provide potential alternative of therapeutic targets and treatments for inflammation-related diseases, and the development of healthier foods and diets.

Graduation Semester

2025-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/129894

Copyright and License Information

Copyright 2025 Jennifer Kusumah

Owning Collections