PMID- 37308226
OWN - NLM
STAT- MEDLINE
DCOM- 20230614
LR  - 20231213
IS  - 2095-4352 (Print)
VI  - 35
IP  - 5
DP  - 2023 May
TI  - [Study on the anti-sepsis mechanism of ursolic acid by targeting myeloid 
      differentiation protein-2].
PG  - 476-481
LID - 10.3760/cma.j.cn121430-20220705-00634 [doi]
AB  - OBJECTIVE: To explore the mechanism of ursolic acid in treating sepsis using 
      myeloid differentiation protein-2 (MD-2) as the research carrier. METHODS: The 
      affinity of ursolic acid and MD-2 was determined by biofilm interferometry 
      technique, and the bonding mode between ursolic acid and MD-2 was tested with the 
      aid of molecular docking technique. Raw 264.7 cells were cultured in RPMI 1640 
      medium and subcultured was conducted when the cell density reached 80%-90%. The 
      second-generation cells were used for in the experiment. The effects of 8, 40 and 
      100 mg/L ursolic acid on cell viability were assessed by methyl thiazolyl 
      tetrazolium (MTT) method. Cells were divided into blank group, lipopolysaccharide 
      (LPS) group (LPS 100 mug/L) and ursolic acid group (100 mug/L LPS treatment after 
      addition of 8, 40 or 100 mg/L ursolic acid). The effect of ursolic acid on the 
      release of cytokines nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and 
      interleukins (IL-6, IL-1beta) were evaluated by enzyme-linked immunosorbent assay 
      (ELISA). The influence of ursolic acid on the mRNA expressions of TNF-alpha, IL-6, 
      IL-1beta, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were 
      detected by reverse transcription-polymerase chain reaction (RT-PCR). The 
      implication of ursolic acid on the protein expressions of LPS-Toll-like receptor 
      4 (TLR4)/MD-2-nuclear factor-kappaB (NF-kappaB) pathway were tested by Western blotting. 
      RESULTS: Ursolic acid could bind to the hydrophobic cavity of MD-2 through 
      hydrophobic bond with the amino acid residues of the protein. Therefore, ursolic 
      acid showed high affinity with MD-2 [dissociation constant (KD) = 1.43x10(-4)]. 
      The cell viability were decreased slightly, with the concentration of ursolic 
      acid increasing, and the cell viability of 8, 40 and 100 mg/L ursolic acid were 
      96.01%, 94.32% and 92.12%, respectively, and there was no significant difference 
      compared with the blank group (100%). Compared with the blank group, the cytokine 
      level of the LPS group was significantly increased. The level of cytokines were 
      significantly reduced by the treatment of 8, 40 and 100 mg/L ursolic acid, and 
      the higher the concentration, the more obvious effect [compared between 100 mg/L 
      ursolic acid group and LPS group: IL-1beta (mumol/L): 38.018+/-0.675 vs. 111.324+/-1.262, 
      IL-6 (mumol/L): 35.052+/-1.664 vs. 115.255+/-5.392, TNF-alpha (mumol/L): 39.078+/-2.741 vs. 
      119.035+/-4.269, NO (mumol/L): 40.885+/-2.372 vs. 123.405+/-1.291, all P < 0.01]. 
      Compared with the blank group, the mRNA expressions of TNF-alpha, IL-6, IL-1beta, iNOS 
      and COX-2 in the LPS group were significantly increased, and the protein 
      expressions of MD-2, myeloid differentiation factor 88 (MyD88), phosphorylation 
      NF-kappaB p65 (p-NF-kappaB p65) and iNOS in the LPS-TLR4/MD-2-NF-kappaB pathway were 
      significantly up-regulated. Compared with the LPS group, the mRNA expressions of 
      TNF-alpha, IL-6, IL-1beta, iNOS and COX-2 were significantly reduced by the treatment of 
      100 mg/L ursolic acid bound with MD-2 protein [TNF-alpha (2(-DeltaDeltaCt)): 4.659+/-0.821 vs. 
      8.652+/-0.787, IL-6 (2(-DeltaDeltaCt)): 4.296+/-0.802 vs. 11.132+/-1.615, IL-1beta (2(-DeltaDeltaCt)): 
      4.482+/-1.224 vs. 11.758+/-1.324, iNOS (2(-DeltaDeltaCt)): 1.785+/-0.529 vs. 4.249+/-0.811, COX-2 
      (2(-DeltaDeltaCt)): 5.591+/-1.586 vs. 16.953+/-1.651, all P < 0.01], and the proteins 
      expressions of MD-2, MyD88, p-NF-kappaB p65 and iNOS in the LPS-TLR4/MD-2-NF-kappaB 
      pathway were significantly down-regulated (MD-2/beta-actin: 0.191+/-0.038 vs. 
      0.704+/-0.049, MyD88/beta-actin: 0.470+/-0.042 vs. 0.875+/-0.058, p-NF-kappaB p65/beta-actin: 
      0.178+/-0.012 vs. 0.571+/-0.012, iNOS/beta-actin: 0.247+/-0.035 vs. 0.549+/-0.033, all P < 
      0.01). However, there was no difference in protein expression of NF-kappaB p65 among 
      the three groups. CONCLUSIONS: Ursolic acid inhibits the release and expression 
      of cytokines and mediators and regulates LPS-TLR4/MD-2-NF-kappaB signaling pathway by 
      blocking MD-2 protein, and thus plays an anti-sepsis role.
FAU - Chen, Guirong
AU  - Chen G
AD  - Institute of Pharmaceutical Research, the 967th Hospital of the Joint Logistics 
      Support Force of the Chinese People's Liberation Army, Dalian 116600, Liaoning, 
      China.
AD  - College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 
      116600, Liaoning, China. Corresponding author: Wang Xiaobo, Email: 
      wxbbenson0653@sina.com.
FAU - Liu, Chang
AU  - Liu C
AD  - College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 
      116600, Liaoning, China. Corresponding author: Wang Xiaobo, Email: 
      wxbbenson0653@sina.com.
FAU - Zhang, Mingbo
AU  - Zhang M
AD  - College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 
      116600, Liaoning, China. Corresponding author: Wang Xiaobo, Email: 
      wxbbenson0653@sina.com.
FAU - Wang, Xiaobo
AU  - Wang X
AD  - Institute of Pharmaceutical Research, the 967th Hospital of the Joint Logistics 
      Support Force of the Chinese People's Liberation Army, Dalian 116600, Liaoning, 
      China.
LA  - chi
PT  - English Abstract
PT  - Journal Article
PL  - China
TA  - Zhonghua Wei Zhong Bing Ji Jiu Yi Xue
JT  - Zhonghua wei zhong bing ji jiu yi xue
JID - 101604552
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 0 (Actins)
RN  - EC 1.14.99.1 (Cyclooxygenase 2)
RN  - 0 (Interleukin-6)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (Lymphocyte Antigen 96)
RN  - 0 (Myeloid Differentiation Factor 88)
RN  - 0 (NF-kappa B)
RN  - 0 (Toll-Like Receptor 4)
RN  - 0 (Cytokines)
RN  - 0 (RNA, Messenger)
SB  - IM
MH  - Humans
MH  - *Tumor Necrosis Factor-alpha
MH  - Actins
MH  - Cyclooxygenase 2
MH  - Interleukin-6
MH  - Lipopolysaccharides
MH  - Lymphocyte Antigen 96
MH  - Molecular Docking Simulation
MH  - Myeloid Differentiation Factor 88
MH  - NF-kappa B
MH  - Toll-Like Receptor 4
MH  - *Sepsis
MH  - Cytokines
MH  - Cell Differentiation
MH  - RNA, Messenger
MH  - Ursolic Acid
EDAT- 2023/06/13 01:13
MHDA- 2023/06/14 06:42
CRDT- 2023/06/12 20:44
PHST- 2023/06/14 06:42 [medline]
PHST- 2023/06/13 01:13 [pubmed]
PHST- 2023/06/12 20:44 [entrez]
AID - 10.3760/cma.j.cn121430-20220705-00634 [doi]
PST - ppublish
SO  - Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2023 May;35(5):476-481. doi: 
      10.3760/cma.j.cn121430-20220705-00634.