The protective effects of leonurine against acute endotoxin induced uveitis in rats / 复旦学报（医学版）

Objective To investigate whether the protective effects of leonurine (SCM-198) against endotoxin induced uveitis (EIU) of SD rats caused by lipopolysaccharide (LPS) was existing,and discuss the underlying mechanisms.Methods Thirty-six normal healthy male SD rats were divided into 3 groups randomly with the same baseline bodyweight and feeding conditions.All rats received intragastric administration every day.The experimental group was devided into 4 subgroups,rats in these subgroups received SCM-198 intragastric administration by as the dose of 10,20,40 and 80 mg/kg bodyweight per day,rats in the negative control group received intragastric administration of normal saline 10 mL/kg per day,rats in the positive control group received intragastric administration ofdexamethasone (DEX) 0.5 mg/kg bodyweight per day.All rats received a 21-day-intragastric administration.The body weight of all rats was monitored every 7 days.The electroretinogram (ERG) examination was taken in the 18th day.All rats received a 100 mg S.typhi LPS intraperitoneal injection after the 21st intragastric administration.Twenty-four hours later,following anaesthesia,all rats received another ERG examination,and inflammation was scoring under microscope by 2 experienced ophthalmologists,after that the aqueous humor of all rats was collected from the left eye.The aqueous humor was kept in-80 ℃ immediately.Then the rats were sacrificed and the right eyes were immediately enucleated to finish the HE staining and immunohistochemistry (IHC) staining examination of tumor necrosis factor-α (TNF-α) and intercellular cell adhesion molecule-1 (ICAM-1).The total amount of protein in aqueous humor was detected by BCA test.Western blot was used to examine the expression of TNF-α,interleukin-1β (IL-1β),IL-6 and ICAM-1.All data was analyzed by SPSS 19.0,and differences were considered significant at P＜0.05.Results The body weight of the rats in positive control group was significantly lower (P＜0.05) than the experimental group and the negative control group after the 21-day-intragastric administration.The inflammatory score of experimental group was lower than that of the negative control group,but higher than the score of positive control group.The HE staining sections showed the similar results.The a wave of ERG in 0.01 cd of rats received 20 mg/kg SCM-198 daily intragastric administration after LPS injection was significantly lower than that before the LPS injection (P＜0.05),also lower than other groups after LPS injection.The expression of TNF-α,IL-6 and IL-1β in the aqueous humor of the rats in the subgroup of SCM-198 10 mg/kg daily intragastric administration was lower than other groups.Conclusions Intragastric administration of SCM-198 has protective effect against endotoxin induced uveitis in SD rats without obvious adverse reaction,which could alleviate the imflammatory reaction and the damage to the uvea construction.NF-κB plays an important role in the reaction.Thus,SCM-198 is a candidate potent compound with potential therapeutic applications in inflammation associated eye diseases.While the best mode and dose of administration should be further investigated.

Experimental autoimmune uveitis and other animal models of uveitis: An update.

Over the past several decades, animal models of autoimmune uveitis directed at eye‑specific antigens (Ags) have been developed. These have allowed researchers to understand the basic mechanisms that lead to these diseases and also recently helped the researchers in translational research for therapeutic interventions. Experimental autoimmune uveitis (EAU) is an animal disease model of human endogenous uveitis and can be induced in susceptible animals by immunization with retinal Ags. Ever since the first description of EAU in mice in 1988, several animal models of uveitis has been described by researchers. Disease‑specific model for cytomegalovirus retinitis and tubercular uveitis has evolved our understanding of these complex entities. Endotoxin induced uveitis is another useful model for anterior uveitis, which is not an autoimmune process and is triggered by injection of bacterial endotoxin (lipopolysaccharides) resulting in a rapid short lasting uveitis. The current article will give an insight into the various EAU animal models and their current implications in translational research. The article will also highlight the different grading systems for EAU in the animal model.

A Novel Therapeutic Target in Inflammatory Uveitis: Transglutaminase 2 Inhibitor

PURPOSE: Our goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU) METHODS: EIU was induced in female Lewis rats by single footpad injections of 200 microgram of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients. RESULTS: Clinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region. CONCLUSIONS: TGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.

Effects of Corneal Neovascularization on TNF-alpha and TGF-beta 2 mRNA Expression in Endotoxin Induced Uveitis

PURPOSE: We tried to evaluate the expression of TNF-alpha and TGF-beta 2 mRNA during the course of the endotoxin induced uveitis (EIU) in rat after inducing neovascularization on cornea. METHODS: Reverse transcription followed by polymerase chain reaction amplification was used to determine relative mRNA level in two ocular tissues (iris-ciliary body, cornea) at 0, 1, 3, 6, 12, 48 hours after subcutaneous injection of 200 ug of Escherichia coli endotoxin. RESULT: Rat with corneal neovascularization showed somewhat different pattern of expression. TGF-beta 2 mRNA expression was showed some fluctuations in cornea. TGF-beta 2 mRNA expression in cornea was decreased within 3 hours after endotoxin treatment and increased gradually after 6 hours until 48 hours. CONCLUSION: TGF-beta2 might be one of the important cytokines in EIU, especially animals with neovascularization on cornea.

The Effect of Heat Shock Protein on Inflammatory Reaction and Cataractogenesis in Endotoxin-induced Uveitis

PURPOSE: Heat shock proteins (Hsps) are expressed by various types of stress, and known to play an important role in protecting cellular homeostasis. We hypothesized that Hsps may protect tissue damage and cataract formation in endotoxin-induced uveitis. METHODS: Lewis rats were exposed to 43degreesC balanced salt solution or arsenite for inducing Hsps, then received LPS injection 6 hours later. We evaluated the inflammatory grade and cataractous lens change. Histological examination, measurement of nitric oxide concentration in anterior chamber and SDS-PAGE analysis of lens protein change were also performed. RESULTS: Western blotting of rat lens revealed that Hsps progressively increased by 6 hour after heating and peaked at 12 hours. The inflammatory grade and cataractous lens change were less in Hsps expressed. Nitric oxide concentration were also lowered in Hsps expressed groups. Analysis of lens protein revealed that normally expressed 66.2 KDa protein disappeared in all groups after LPS injection but recovered in Hsps expressed groups with time. CONCLUSIONS: This result suggests that Hsps may protect tissue damage and cataract formation in endotoxin-induced uveitis. This effect may be associated with decreased nitric oxide. The study demonstrates that preconditioning to induce larger amount of Hsps by heat shock or drug may decrease inflammation and cataractogenesis induced by uveitis and surgery. We conclude that induction of Hsps may be useful as a new therapeutic modality against uveitis-induced cataract.

The Reversible Y-Suture Lens Opacity Formation in Endotoxin Induced Uveitis Model

The present study was undertaken to find out the role of NO on cataractogenesis in the experimenally-induced uveitis(EIU) model. Nitrite and nitrate, stable oxidative products of nitric oxide(NO), were measured in the aqueous humor and the progression of inflammations and lens opacities were evaluated with slit lamp biomicroscope. Immunoperoxidase staining and immunofluorescent staining for inducible NO synthase(iNOS) and peroxynitrite were performed to confirm the site of production of NO and peroxynitrite. The grades of inflammation were peaked at 24 hours inflammation was gradually decreased after 48 hours and lens opacity after 72 hours. These changes returned to the baseline level by one week after LPS injection. Similarly, NO concentration in aqueous humor was peaked at 24 hours. And it was then decreased after 48 hours and returned to the baseline level by one week. These inflammatory signs and lens opacities were significantly decreased in NG-nitro-L-arginine methyl ester(L-NAME) administrated group. Inflammatory cells in anterior chamber, iris, and ciliary body expressed highly iNOS which was coincide with peroxynitrite immunolocalization. Therefore, these results suggest that cataract formation in EIU is related to the NO production in aqueous humor. Furthermore, lipid peroxidation by peroxynitrite is possibly related with cataractogenesis in EIU. But, we need a further evaluation to seek the relationship between cataractogenesis and increased nitric oxide concentration, combined with studies of other biochemical changes in anterior chamber and lens.

Study of Pro-inflammatory Cytokines in Endotoxin Induced Uveitis

Cytokines are produced during the effector phases of natural and specific immunity and serve to mediate and regulate immune and inflammatory responses. In natural immunity,endotoxin directly stimulates mononuclear phagocytes to secrete their cytokines and cytokines which were produced by one cell type often regulate the synthesis of cytokined by other cells. To gain effects on the role of pro-inflammatoy cytokines(TNF, IL-1, IL-6) in the endotoxin induced uveitis, author investigated the cell counts of infilterating inflammatory cells, IL-6 bioassay in aqueous humor, mRNA analysis in the transgenic endotoxin induced uveitic mice(TNF-R(-), IL-1R(-), TNF-R(-)/IL-1R(-)). In this study, there was no significant difference in the number of infiltering cells in TNF-R(-) mice compared to congenic controls, but significant diffetence in the unmber of infiltering cells was found in IL-1R(-) and TNF-R(-)/IL-1R(-) mice. IL-6(-) levels in aqueous humor were reduced in IL-1R(-) and TNF-R(-)/IL-1R(-) mice. TNF-alpha, IL-1alpha, IL-6 mRNA were detected in all experimental mice after endotoxin injection. IL-1alpha and IL-1Ralpha mRNA were detected in noninjected TNR-R(-) mice. IL-1beta mRNA was not detected in all experimental mice without or with endotoxin injection. In conlclusion, IL-1 appears to have a more important role in endotoxin induced uveits than TNF. Though IL-6 is detected in endotoxin induced uveitis, it is not an indicator for the enhenceing inflammation and may not be essential for the endotoxin induced uveitis.

Distribution of MHC class II Positive Cells and Macrophages in the Iris and Ciliary Body in Endotoxin Induced Uveitis

Acute anterior uveitis was induced by footpad injection of bacterial lipopolysaccharide(LPS) in the Lewis rats. To evaluate the distribution and density of MHC class II positive cell, macrophages, B lymphocytes infiltration in the iris and ciliary body 24 hours after injection of bacterial LPS, immunohistochemical study was performed in the wholemount tissues with monoclonal antibodies. Quantitative analysis reveals that the density of MHC class II positive dendritic cells in the iris was 348.5+/-55cells/mm2 in the control group and 448.0+/-176cells/mm2 in the LPS injected group(p<0.05). The density of ED2 positive resident tissue macrophages was higher in the LPS injected group(761.9+/-82cells/mm2) than the control group(620.8+/-57cells/mm2)(p<0.05). ED1 positive macrophages infiltrated significantly in the LPS results in increased group(3225.0+/-522cells/mm2) than in the control group(590.5+/-52cells/mm2).OX33 positive cells were not observed in both control and LPS injected eyes. In conclusion, destruction of blood ocular barrier by infection of LPS results in increased infiltration of OX6 positive cells and macrophages, particularly massive influx of blood monocytes into the iris and ciliary body in acute phase of inflammation. This study confirmed that dendritic cells and macrophages play important roles in acute phase of endotoxin induced uveitis.

Non-steroidal anti-inflammatory drug and endotoxin induced uveitis

Suprofen eye drop was instilled into one eye of 10 pigmented rabbits and then anterior uveitis was induced by intraperitoneal injection of endotoxin of Shigella flexneri serotype 1A to evaluate the effects of non-steroidal anti-inflammatory drug on endotoxin induced uveitis. The pupillary diameters were measured, and aqueous cell and flare gradings were recorded in 20 eyes of 10 rabbits for one week at an interval of 12 hours for the first 24 hours and then every 24 hours for a week. A difference between the treated and control groups were investigated. All the above parameters showed greatest changes at 12 or 24 hours after injection and became normal by one week. The two groups demonstrated statistically significant difference at 12 hours, day 1 and day 2 as for pupillary diameter, at day 1 and day 2 as for cell and at 12 hours and day 1 as for flare. Thus, it can be concluded that prostaglandins play a role in miosis, in the appearance of inflammatory cells and flare in endotoxin induced uveitis and the topical administration of non-steroidal anti-inflammatory drug can alleviate signs of anterior uveitis. Specific relationship between leukotriene B4 and aqueous cell was not demonstrated.