| IntroductionErythromycin is a 14 -member lactone ring macrolide antibiotic which has long been used for the treatment of infectious diseases. Apart from its antibacterial activity, erythromycin exhibits a broad spectrum of pharmacological effects, including anti - inflammatory. Long - term, low - dose erythromycin therapy is effective in the treatment of diffuse panbronchiolitis ( DPB). It is believed that erythromycin produce this benefit through an anti - inflammatory effect rather than antibacterial effect. Beside DPB, erythromycin is known to be effective for the treatment of other chronic inflammatory airway diseases including chronic bronchitis and bronchial asthma. Although there were numerous reports in the past few years of macrolide antibiotics having anti - inflammatory activity, little attention had been paid to using them for the treatment of other inflammatory diseases.Rheumatoid arthritis ( RA) is a chronic polyarthritis characterized by the o-vergrowth of synovial fibroblast and inflammatory cell infiltration. This invasive growth of synovial tissues corresponds with the progressive destruction of articular carilage and bone leading to remarkable disability. Infiltrated T lymphocytes and synovial fibroblasts from patients with rheumatoid arthritis express the receptor activator of NF - kB ligand ( RANKL) , which appears to trigger bone destruction in rheumatoid arthritis. This raises the possibility that RANKL therapy to block this mechanism might prove beneficial in patients with RA. Recent studies have shown that T lymphocytes produce cytokines other than RANKL such as IL - 17, granulocyte macrophage colony - stimulating factor ( GM -CSF) and IFN --γ, which have powerful regulatory effects on osteoclastogene-sis. Hence, the role of T lymphocytes in the pathogenesis of RA at a chronic stage has attracted attention.Nuclear factor kappa B ( NF - kB) is a nuclear transcription factor ubiquitously involved in gene expression for a variety of proi nfl ammatory cytokines. NF - kB is in an inactive form in the cytoplasm. During the activation, NF - kB is released from IkB through the phosphorylation and degradation of IkB. Activation of NF - kB can induce to transcription of many cytokine genes and inhibit apoptosis in inflammatory responses. Meanwhile, activation of RANKL results in the expression of NF - kB.Considering both RANKL and NF - kB that are secreted by T lymphocytes play very important roles in the inflammatory response, we carried out a series of experiments to evaluate the effects of erythromycin and its derivatives without antibacterial activity on proliferation of T lymphocyte and expression of RANKL and NF - kB.Methods1. T lymphocyte was grown in RPMI 1640 medium containing 10% FBS and 100 U of penicillin and 100jxg of streptomycin/mL at 37°C under humidified 5% CO2 as stationary cultures. T lymphocyte was treated with EM and nine derivatives at a dose range of 3. Ojig/mL to 100. Ojxg/mL for 24h. Then the inhibition of T lymphocyte proliferation induced by erythromycin and its derivatives was examined by MTT assay2. T lymphocyte was pretreated with EM and two derivatives I and VIIII at a dose range of 3. Ojig/mL - 100. Ojxg/mL for 24h. Flow cytometric analysis was performed by PI staining. T lymphocyte apoptosis and cell cycle were detected by flow cytometry. In the meantime, T lymphocyte apoptosis was confirmed by TUNEL staining. The optical density was analyzed by micro - image analysis system.3. T lymphocyte was exposed to 100 pM TNF - a with or without pretreat-ment with 3.0,10.0,30.0 or 100.0u,g of EM, I and VIII/mL 1 h at 37°C. The expressions of RANKL and NF - kB were observed by RT - PCR and WesternBlot. The intensity of expression was analyzed by micro - image analysis.4. Statistics: Data were expressed as Mean ± SD. Statistical analysis was performed with one - way analysis of variation ( ANOVA) and correlation. P < 0.05 was considered significant.Results1. MTT assay showed that EM, CAM, I, IV and VIII could inhibit T lymphocyte proliferation. Their IC^ values were 606.28 ± 35.43, 431. 62 ± 25.60, 367.31 ±32.60, 425.62 ±32.08, 176.38 ± 10.42uJVI, respectively. The IC^ values of I and VIII were significantly lower than those of EM and CAM.2. Flow cytometry and TUNEL revealed that EM (30. Ojxg/mL - 100. Ojxg/ mL) , I (3. Ojig/mL -30. 0|xg/mL) and VIII (30. Ojig/mL) could induce T lymphocyte apoptosis. VIII (3.0(xg/mL -30.0(xg/mL) arrested T lymphocyte cell cycle progression in G2/M phase in a concentration - related manner. I and VIII induced cell to die at 100.0|xg/mL.3. Western blot analysis of nuclear extracts of T lymphocyte stimulated with 100 pM TNF - a for lh revealed that pretreatment with EM, I and VIII decreased the expressions of NF - kB protein and RANKL protein in a dose - related manner. The treatment of EM (100. O^g/mL) , I (100.0u,g/mL) and VIII (100. OjjLg/mL) resulted in about 46% , 59% and 36% reduction of NF - kB protein, meanwhile, 32% , 40% , 20% reduction of RANKL protein. The expression of NF - kB protein correlated with the expression of RANKL protein.4. RT - PCR analysis showed that EM, I and VIII decreased the expressions of NF - kB mRNA and RANKL mRNA in a dose - related fashion. The treatment of EM (100.0|xg/mL) , I (100. Ou.g/mL) and VIII (100.0|Ag/mL) led to 36% , 46% and 32% reduction of NF - kB mRNA, and 29% , 37% , 24% reduction of RANKL mRNA. The expression of NF - kB mRNA was correlation with the expression of RANKL mRNA.ConclusionErythromycin and its derivatives could inhibit T lymphocyte proliferation and expression of NF - kB and RANKL. Taken together, these data suggest EM and its derivatives have an anti - inflammatory activity, presumably via an interaction with the RANKL signaling pathway, resulting in the inhibition of NF - kB , along with apoptosis and cycle arrest of T lymphocyte. This is the first study to show that the key role of RANKL is involved in anti - inflammatory functions of erythromycin and its derivatives. Our findings provide a novel mechanism for the anti - inflammatory activity of macrolides, implicating a target for the development of new drugs for treating, such as RA, inflammatory diseases. |
