Abstract
Purpose.:
The purpose of this study was to explore the effect of artesunate on endotoxin-induced uveitis (EIU) in rats.
Methods.:
EIU was induced in male Long-Evans rats by footpad injection of lipopolysaccharide (LPS 200 μg in 0.1 mL PBS; LPS-only group [LG]). PBS (0.1 mL), prednisolone (10 mg/kg in 0.1 mL PBS; prednisolone group [PG]), and artesunate of three concentrations (1, 10, 100 mg/kg; artesunate groups A, B, C [AGA, AGB, AGC]; all in 0.1 mL of PBS) were injected intravenously, respectively, 1 hour after LPS injection. Twenty-four hours after LPS injection, infiltrating cells and protein concentration in the aqueous humor, as well as tumor necrosis factor (TNF)-α, nitric oxide (NO), prostaglandin E2 (PGE2), and monocyte chemoattractant protein (MCP)-1, were measured.
Results.:
Rats in AGA did not have significant difference in infiltrating cell number (61.6 × 105 cells/mL; range, 20.0–147.8 cells/mL; P = 0.287), protein concentration (48.0 mg/mL; range, 18.0–101.8 mg/mL; P = 0.349), NO concentration (86.9 μM/L; range, 30.5–192.5 μM/L; P = 0.363), but had significant difference in TNF-α (1144.3 pg/mL; range, 460.0–1840.0 pg/mL; P = 0.038), PGE2 (12.8 ng/mL; range, 4.8–27.2 ng/mL; P = 0.005), and MCP-1 (6136 pg/mL; range, 20.0–147.8 pg/mL; P = 0.009) with those of LG. Artesunate at 10 mg/kg and 100 mg/kg significantly suppressed the infiltrating cells, protein concentration, TNF-α, NO, PGE2, and MCP-1 in the aqueous humor induced by LPS (P = 0.011–0.000).
Conclusions.:
Dose-dependent decreases of infiltrating cells, protein concentration, TNF-α, PGE2, NO, and MCP-1 in the aqueous humor by artesunate treatment after LPS injection indicate that artesunate can suppress the inflammation of EIU by inhibiting the production of inflammatory mediators.
Endotoxin-induced uveitis (EIU) is an acute uveal inflammation that is induced by intraocular injection of lipopolysaccharide (LPS).
1 It has been reported that EIU could be regarded as an experimental model for some types of human uveitis, which may be connected with Reiter's syndrome, where Gram-negative bacteria such as
Shigella,
Salmonella, and
Yersinia may play an important role.
2 The inflammation of EIU is characterized by the infiltration of iris and ciliary body by a large number of inflammatory cells, accompanied with cellular exudation into the anterior chamber. Exposure to LPS stimulates cellular inflammatory responses, and releases inflammatory mediators such as cytokines and chemokines, including tumor necrosis factor alpha (TNF-α),
3 interleukin-6 (IL-6),
4 and monocyte chemoattractant protein 1 (MCP-1).
5
The metabolites of arachidonic acid such as prostaglandin-E2 (PGE2) are now well appreciated. These metabolites could be found at inflammatory sites and lead to local blood flow increases, edema formation, and pain sensitization in the aqueous humor. Furthermore, the generation of reactive oxygen species (ROS) and excessive amounts of nitric oxide (NO) are other responses.
6 In the present study, we investigated eye inflammation by measurement of infiltrating cells, protein concentration, PGE2, TNF-α, NO, and MCP-1 levels in aqueous humor.
Artesunate, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb artemisia annua, is a safe and effective antimalarial drug.
7 Artesunate has been reported to possess antitumor
8,9 and anti-angiogenic effects,
10 to decrease serum endotoxin release in sepsis mice,
11 and to have a protective effect on LPS-induced human umbilical vein endothelia-1 cell (HUVEC) activation, which might be associated with the inhibition of TNF-α mRNA expression and a decrease in the secretion of interleukin (IL)-1 beta, IL-6, and IL-8 from TNF-α stimulated rheumatoid arthritis (RA) or fibroblast-like synoviocytes (FLS).
12
However, whether artesunate is capable of reducing inflammatory cytokine release of EIU in vivo is unknown. In this report, we investigated the effects of artesunate on endotoxin-induced uveitis in rats in vivo and examined the possible molecular mechanisms involved in the inhibition of inflammatory responses released by artesunate.
In the present study, we investigated the effect of artesunate on EIU in rats. Decreases of infiltrating cells, protein concentration, TNF-α, PGE2, NO, and MCP-1 contents in the aqueous humor by artesunate treatment after LPS injection indicate that artesunate can suppress the inflammation of EIU by inhibiting the production of inflammatory mediators dose-dependently. Its anti-inflammatory effect on EIU was comparable to the effect of prednisolone used in similar dose.
We chose the infiltrating cells number and protein concentration in the aqueous humor as the indicators of the degree of anterior inflammation. We found artesunate at 10 mg/kg and 100 mg/kg significantly suppressed the infiltrating cells and protein concentration. The exact mechanism by which artesunate inhibits EIU is yet to be studied; however, our results support the findings that artesunate suppresses EIU by blocking the local TNF-α production. TNF-α is a cytokine involved in systemic inflammation, cell apoptosis, cell death, and immune cell regulation. It is a member of a group of cytokines that stimulate the acute phase reaction of inflammation.
13 In this study, TNF-α concentrations in the aqueous humor of artesunate treated groups were significantly decreased. In the report of Gumede,
14 artesunate produced dose-dependent inhibition of ConA-induced TNF-α release of splenocytes from healthy C57BL/6 mice in an in vitro system, which is in accordance with our results.
Nitric oxide is generated by phagocytes (monocytes, macrophages, and neutrophils). It is well known that oxidative stress is the major factor in the course of local inflammation and tissue damage during inflammatory process. In the human immune response, phagocytes are armed with inducible nitric oxide synthase (iNOS), which is activated by tumor necrosis factor. NO plays an important role in endotoxemia and inflammatory conditions.
15 As a member of oxidative stress factors, nitric oxide secreted is toxic to cells for DNA damage and degradation of iron sulfur centers into iron ions and iron-nitrosyl compounds were found.
16 In this study, elevated levels of nitrite would be related to increased NO generation in the inflamed eye because NO is rapidly oxidized to nitrite and then to nitrate and the Griess reagent can only assay for nitrite. Also, some of the NO can combine with reactive oxygen to become peroxynitrite, which is also rapidly lost. Our results indicated that artesunate at 10 mg/kg and 100 mg/kg suppressed the NO level (actually, level of nitrite) in the aqueous humor induced by LPS significantly. The results of this study showed that artesunate suppresses the expression of NO in EIU rats.
PGE-2 is one of the prostaglandins, a group of hormone-like substances involved in various functions such as contraction and relaxation of smooth muscle, dilation and constriction of blood vessels, blood pressure control, and inflammation modulation.
17 When tissues are exposed to different physiological and pathologic stimuli, arachidonic acid is liberated from membrane phospholipids by phospholipase A2 and is transformed to PGH2 by prostaglandin H synthase (PGHS; also known as cyclooxygenase [COX]). PGH2 is the common substrate for PGD2, PGE2, prostacyclin (PGI2), and tromboxane (TXA2). PGE2 plays important roles in various biological activities such as neuronal function, female reproduction, vascular hypertension, tumor angiogenesis, renal function, and inflammation. In our study, artesunate suppressed PGE2 levels markedly. Artesunate at 1 mg/kg, 10 mg/kg, and 100 mg/kg significantly suppressed PGE2 in the aqueous humor induced by LPS.
MCP-1 recruits monocytes, memory T cells, and dendritic cells to sites of tissue injury and infection.
18,19 It is a monomeric polypeptide located on chromosome 17 in humans, with a molecular weight of approximately 13 kDa. MCP-1 has monocyte activation, demonstrated in human eyes during acute anterior uveitis and described in the rat EIU model.
20 Data of this experiment showed that artesunate can decrease MCP-1 concentration in the aqueous humor, suggesting that artesunate expresses its anti-inflammatory action by affecting monocytes, or memory T cells, or dendritic cells recruitment.
Since endotoxin-induced uveitis is the result of a systemic response to an endotoxin injection distant to the eye and that the artesunate itself was systemically injected. The results of this study showed that artesunate has a significant effect on endotoxin-induced inflammation, which then suppresses EIU.
Disclosure:
X.-Q. Wang, None;
H.-L. Liu, None;
G.-B. Wang, None;
P.-F. Wu, None;
T. Yan, None;
J. Xie, None;
Y. Tang, None;
L.-K. Sun, None;
C. Li, None