| Human cytomegalovirus (HCMV), a double-stranded DNA virus in the herpes virus family, is widespread opportunistic pathogen that causes acute, latent, and chronic infections in the world. Our country is a high-risk area in HCMV infection. Most of the primary infections occurred in children under2years old, and children’s anti-HCMV serum antibody positive rate is83.2-87.3%, while adult’s as high as95%. But most patients have no clinical signs or symptoms. CMV infection can be latent in the salivary glands, mammary glands, kidney and leukocyte for a long time. When the body’s immune function is low, the virus could be activated and lead to many diseases. CMV could cause several oral diseases, such as sialadenitis, Sjogren’s syndrome, periodontitis, recurrent aphthous ulcer and salivary gland tumors, seriously affecting people’s physical and mental health. Domestic and overseas scholars have already researched on CMV pathogenic mechanism, prevention and treatment measures. We realized cellular immunity play an important role in limiting CMV transmission and reactivation. CD81T lymphocytes play a key anti-viruses role in CMV infections. In addition, in the acute phase of CMV infection, the body produced corresponding humoral immunity by virus stimulation. Secretory IgA participates in local mucosal immune, and plays an important role in the resistance of CMV infection.Chemokines are known to play pivotal roles in innate and acquired immunity by regulating migration and activation of leukocytes via a group of seven transmembrane G protein-coupled receptors. Chemokines are small (8-16kDa), and have20-70% amino acid homology of protein family. According to the arrangement of the amino-terminal conserved cysteine residues, the chemokines are classified into four subfamilies:CXC, CC, C, and CX3C. CCL28(also called mucosae associated epithelial chemokine) is a recently described CC chemokine. The transcripts are detected in a variety of tissues but most abundantly in trachea, colon, rectum, and exocrine glands such as salivary and mammary glands. CCL28attracts cells(CD4+T lymphocyte, CD8+T lymphocyte, IgA antibody secretory cells, eosinophils) expressing its receptors CCR10and CCR3into certain mucosal tissues. Accumulating evidence points out that chemokines and antimicrobial peptides have substantially overlapping functions. CCL28exerts a potent and salt-sensitive antimicrobial activity against a broad spectrum of microbes including Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. CCL28works with other mucosa antimicrobial factors to constitute a broad-spectrum antimicrobial cover. More important, CCL28play an important role in immune response by regulating migration and activation of CD8+T lymphocyte and IgA antibody secretory cells via its receptors CCR10and CCR3.Nuclear transcription factor-KB(NF-KB) is a kind of multipolarity gene regulation protein, exists in the form of the same or different dimmers constituted by the Rel protein family members. NF-κB could be found in the almost all the cells. So far, we have found a variety of factors(Viruses, LPS, double-stranded RNA, etc.; Inflammation cytokines; Physical and chemical factors; Apoptosis factors) that can induce NF-κB activation to start the transcription of the corresponding genes to regulate the expression of some important cytokines, inflammation medium, adhesion molecules and protein enzymes. Therefore. NF-κB, who has a pivotal position in the immune system, is the regulated hub of many corresponding gene transcription to control the body’s immune status.Accumulating evidence points out that a variety of cytokines or protein are regulated via NF-κB signal, including①cytokines and growth factors:IL-2,3,6,8,12, IL-1β, TNFα, G-CSF, M-CSF, GM-CSF, EPO, etc;②mmunologic Receptors: immunoglobulin κ light chain, T cell receptor α,β chain, MHC-Ⅰ, Ⅱ, β2micro globulin, etc.③dhesion molecule:Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, etc.;④the acute phase proteins:angiotensin enzyme, complement factor C4, complement factor B, etc.;⑤inflammatory enzyme:Inducible nitric oxide synthase, inducible cyclooxygenase, oxygen enzyme, etc. So far, there is no report about the CCL28expression in vivo.Purpose:In this study, the BALB/c mice model system of MCMV infection was established. The submandibular glands were dissected successfully. We detected the CCL28expression in the submandibular glands at different time points. In order to further reveal the related signal transduction pathways, PDTC pretreatment inhibited NF-κB activation to further observe the change of CCL28expression. Based on the experimental results, the relationship between CCL28and NF-κB was analyzed. This may explore a new related signaling pathway for the treatment of oral diseases with CMV infected.Methods:1. MCMV was inoculated in the3T3cells. When80%-90%cells occured cytopathogenic effect, MCMV was collected and tested the virus titer. According to the data in the literature, BALB/c mice was intraperitoneally injected MCMV. Animal models were proved to be successfully established by enzyme-linked immunosorbent assay(ELISA), and inflammation infiltration situation of the submandibular gland were observed by hematoxylin-eosin (HE) staining.2. The submandibular glands of normal and MCMV infected BALB/c mice were dissected on the7th or15th day, respectively. The level of CCL28mRNA and protain was detected through real-time fluorescent quantitative PCR and Western blotting respectively.3. BALB/c mice were randomly divided into three groups:Group a was blank control group; the mice of group b were injected200μ15×104PFU MCMV; the mice of group c were injected PDTC every day to inhibite the activation of NF-κB after MCMV injected. The submandibular glands were dissected on the7th day. CCL28expression was detected through real-time fluorescent quantitative PCR and Western blotting; the activation of NF-κB was detected by Western blotting. Based on the experimental results, the relationship between CCL28and NF-κB was analyzed.Results:1.3T3cells appears CPE after MCMV infected, the virus titer was4.68×106TCID50. The serum samples were detected for antiMCMV-IgM by ELISA, the results suggested that the BALB/c mice model infected by MCMV was successfully established. The pathological sections showed that in submandibular glands there were lymphocytes and monocytes infiltration around the duct, moreover duct degeneration could be observed.2. CCL28mRNA and protein expression in BALB/c mice infected by MCMV on the7th day in submandibular glands were significantly higher than the normal group; but in the group of the15th day that was slightly increased compared with normal group(no statistical significance), and decreased siginificantly compared with the group of the7th day(p<0.05) with statistical signifance.3. Western blotting suggested the activation of NF-κB were inhibited after injected PDTC compared with only MCMV infected. The expression of CCL28was also decreased obviously after inhibiting the activation of NF-κB.Conclusion:1. The BALB/c mice model system of MCMV infection was successfully established, and its pathological sections showed the typical pathological changes.2. The CCL28expression in the submandibular gland of BALB/c mice infected by MCMV increased significantly in the acute phase, we consider the mechanism was MCMV replicated more to induce the activation of transcription factor related with CCL28. As CCL28expression increased, immune cells (CD4-T lymphocyte, CD8+T lymphocyte, IgA antibody secretory cells, eosinophils) expressing receptors CCR10and CCR3were attracted into certain mucosal tissues to regulate the local immune response. Leading to that the activation of transcription factor was weakened. So CCL28expression went back to the baseline after the acute phase.3. CCL28expression in submandibular glands was reduced significantly compared with that in the mice only infected by MCMV. PDTC could effectively inhibit the activation of NF-κB. Consistent with this, CCL28expression was reduced significantly. That proved NF-κB plays a direct or indirect role in CCL28expression. CCL28may be used in anti-CMV infection by its chemotatic function. It may provide a new way for the MCMV tratement. |
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