| Background and aimsSjogren's syndrome(SS)is a common and chronic systemic autoimmune disease.The predominant pathological manifestation is the infiltration of a large number of lymphocytes in the exocrine glands,especially the T lymphocytes and a vatiety of cytokines,resulting in the dysfuction of the affected glands.Patients often suffer from the dryness of the mouth,eyes,skin and vagina.In addition to the local symptoms,the respiratory,blood,nervous and other systems can also be involved,which seriously affect the quality life of patients.At present,the treatment of SS is mainly aimed at alleviating the symptoms,preventing the development of the disease and prolonging the survival time of the patients,which includes the symptomatic treatment of xerostomia and xerophthalmia,and the use of glucocorticoids,and immunosuppressive agents(cyclophosphamide,azathioprine)when the important organs are involved.Since these medications can not cure SS fundamently and have side effects,the new potent agents without toxicity are urgently needed.Carboxyamidotriazole(CAI)is a non-cytotoxic antitumor drug in development.Recently,we found that CAI also has the considerable anti-inflammatory potency in a variety of animal models of acute and chronic inflammatory diseases and autoimmune diseases.Especially,the anti-inflammatory effect of CAI was associated with its ability to decrease the levels of proinflammatory cytokines.In these inflammatory animal models,CAI can inhibit the activation of NF-?B signaling pathway,and reduce the levels of proinflammatory cytokines in the inflammatory sites and the serum.Since the activation of NF-?B pathway and the increase of cytokines play the important role in the pathological process of SS,we evaluated the therapeutic effects and possible mechanisms of CAI on SS in non-obese diabetic mice(NOD mice)model with spontaneous SS.In addition,considering that the glandular epithelial cells are both the target cells of immune damage and also the important participant of inflammation delay and glandular injury in the pathogenesis of SS,we established the methods of isolation and culture of the submandibular gland epithelial cells of mouse,and explore the effects of CAI on the glandular epithelial cells in vitro.MethodsThe NOD mice with the spontaneous SS were used,which were divided into PEG 400 group,CAI low dose group(20 mg/kg),CAI high dose group(40 mg/kg)and positive drug(total glucosides of paeony,TGP)group.And ICR mice were used as normal control group.The mice were administrated the corresponding drugs from 4 weeks of age and continued to 20 weeks of age.The weight of mice,water intake,saliva flow rate,blood sugar level,and the weight of the submandibular gland,spleen and pancreas were measured.The levels of autoantibodies(anti-SSA/Ro antibody and anti-SSB/La antibody)in the serum were determined by enzyme-linked immunosorbent assay(ELISA).And the pathological changes in the submandibular gland and pancreas were detected by tissue section and hematoxylin and eosin(HE)staining.The contents of IL-1? IL-4 and CXCL1/GRO-?(melanoma growth-stimulatory activity/growth regulated protein-a)in the submandibular gland were tested by eletrochemilunescence(ECL),and the expressions of NF-?B p65 and I?B? in the submandibular gland were assessed by immunohistochemical staining.Collagenase type ? was used to digest and isolate the submandibular cells of mice in vitro.After purified by differential attachment method,trypan blue staining was employed to determine the cell survival rate,optical microscope was utilized to observe the cell morphology,immunofluorescence staining was conducted to identify the cells,and the cell proliferation was detected by CCK-8 method.The cells were stimulated by lipopolysaccharide(LPS)or TNF-? with or without CAI at the concentrations of 20,40 ?mol/L.And the content of IL-6 in the culture supernatant was determined by ELISA method.Results1.The water intake of PEG 400 group began to increase significantly from 11 weeks of age compared with that of Control group(P<0.05),and the increasing extent increased distinctly with the growth of the week age.However,the water intake of CAI(20 and 40 mg/kg)groups increased from 14 weeks of age(P<0.05),which suggested that CAI could delay the onset time of increase of the water intake in NOD mice.In addition,compared with PEG 400 group,CAI(20 and 40 mg/kg)began to relieve the symptoms of increased water intake in NOD mice from 16 weeks and 15 weeks of age(P<0.05 and P<0.01),respectively.2.The saliva flow rate of mice was measured at 12 and 20 weeks of age.The results showed that the saliva flow rate of PEG 400 group was significantly decreased at both time points(P<0.01).While the administration of CAI(20 and 40 mg/kg)could increase the saliva flow rate of mice.When compared the saliva flow rate between 12 and 20 weeks of age,the mice of PEG 400 group at 20 weeks of age showed lower saliva flow rate than 12 weeks of age(P<0.01).However,the salivary flow of CAI(20 and 40 mg/kg)group at 20 weeks of age was evidently higher than that of 12 weeks of age(P<0.05 and P<0.01).These results suggested that CAI could improve the secretion dysfunction of salivary glands in NOD mice.3.The levels of anti-SSA/Ro and anti-SSB/La antibodies in the serum of PEG 400 group were significantly higher than those in Control group(P<0.01).Compared with PEG 400 group,the levels of the two autoantibodies in the serum of CAI(20 and 40 mg/kg)groups were evidently decreased(P<0.05 and P<0.01).4.The weights of submandibular gland and spleen in PEG 400 group were significantly lower than that in Control group(P<0.01).The administration of CAI(20 and 40 mg/kg)could increase the weight of the submandibular gland(P<0.05 and P<0.01),but had no effect on the weight of the spleen.5.The histopathology of PEG 400 group showed that a large number of lymphocytes infiltration,incomplete or damaged acinus,the glandular atrophy and the significantly elevated histologic score(P<0.01),which was similar to the pathological changes of human SS.CAI(20 and 40 mg/kg)could improve the above lesions,showing decreased lymphocytic infiltration,alleviated acinic damage and the obviously lower histological score(P<0.01).6.The pancreases were weighed and histologically tested,and the results showed that,the weight of pancreas in PEG 400 group was obviously decreased(P<0.01)and the histopathological damage was slight.CAI treatment had no effect on the weight of pancreas(P>0.05),nor did it improve the histopathological injury of the pancreas.In addition,there was no correlation between the salivary secretion and the blood sugar level in NOD mice(P>0.05).These results suggested that the SS in NOD mice was not related to its spontaneous insulin dependent diabetes mellitus,and the relief of the SS symptoms in NOD mice by CAI had nothing to do with the diseased pancreas.7.Compared with Control group,the contents of IL-1? IL-4 and CXCL1/GRO-a in the submandibular gland homogenate of PEG 400 group were significantly increased(P<0.01).CAI(20 and 40 mg/kg)could reduce the levels of the above factors(P<0.05 and P<0.01).8.The immunohistochemical result of NF-?B p65 showed that the expression of NF-?B p65 in the submandibular gland of Control group was in a low level and mainly located in the cytoplasm,and the staining score was low.The expression of that in PEG 400 group was much higher than that of Control group and particularly located in the cell nucleus with the marked increase in staining score(P<0.01).The administration of CAI(20 and 40 mg/kg)could significantly decrease the staining of NF-?B p65 both in the cell nucleus and in the cytoplasm,and reduce the staining score(P<0.01).These results indicated that CAI could inhibit the activation of NF-?B in the submandibular gland of NOD mice.9.The immunohistochemical result of I?B? showed that the staining level of I?B?in the submandibular gland of PEG 400 group was lower than that of Control group,and the staining score was also obviously decreased(P<0.01).The administration of CAI(20 and 40 mg/kg)could enhance the staining of I?B? and increase the staining score significantly(P<0.01),which suggested that CAI could inhibit the degradation of I?B? in the submandibular gland of NOD mice.10.The submandibular gland epithelial cells could be successfully obtained by collagenase digestion.The cell survival rate was 97.5%.The morphology characteristic showed the typical epithelioid with polygon in the arrangement of typical "pebble stone" appearance.The cells were stable in growth with active proliferation according to the proliferation curve and could be subcultured to 3 passages.Immunofluorescence results showed that the expression of cytokeratin 8 was positive while vimentin was negative,which was consistent with the phenotypic characteristics of salivary gland cells.11.Incubation by CAI(20 and 40 ?mol/L)for 24 h had no effect on the viability of the submandibular gland epithelial cells.LPS or TNF-? significantly increased the level of IL-6 in the culture supernatant(P<0.01),and CAI(20 and 40 ?mol/L)exhibited the inhibitory effect on the increased level of IL-6 induced by the above stimulants(P<0.05 and P<0.01).ConclusionsCAI can delay the onset of spontaneous in NOD mice and exhibits the fine improvement on the manifestations of SS,which includs that reducing the amount of water intake,increasing the saliva flow rate,decreasing the levels of anti-SSA/Ro and anti-SSB/La antibodies in the serum,increasing the weight of the submandibular gland,and improving the histological injury of the submandibular gland.CAI has no effect on the pancreatic lesion in NOD mice,which indicated that the improvement effect of CAI on SS symptoms in NOD mice is not achieved by alleviating pancreatic lesions.The possible mechanism of CAI on SS might be related to inhibiting the activation of NF-?B signaling pathway,blocking the actions of autoantibodies,reducing the levels of IL-1?,IL-4 and CXCL1/GRO-a in the submandibular gland,and reducing the effects of cytokines network on the submandibular gland epithelial cells. |
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