| Objective:To investigate the effet of chitosan on rabbit carotid artery internal jugular veinfistula intimal hyperplasia. Whether TLR4/NF-κB pathway may play an role inchitosan intervening rabbits AVF.Methods:In vivo:21New Zealand white rabbits were randomly divided into3groups,including normal control group（n=3）, model group（n=9）and chitosan group（n=9）.Model group and chitosan group were established respectively carotid artery internaljugular vein fistula models. After AVF surgery, chitosan was smeared on venousblood vessels and anastomosis. After4,6and8weeks, they were separately sacrificedand taken AVF venous vascular tissue. Labeled group A (normal control group),group B (4weeks model group), group C (4weeks chitosan group), group D (6weeks model group), group E (6weeks chitosan group), group F (8weeks modelgroup) and group G (8weeks chitosan group). Observed pathological changes ofAVF venous vascular tissue in each group. The changes of a-SMA was detected byimmunohistochemistry method. The mRNA expression of PCNA and TLR4in thosetissues were measured by Real-time PCR. At the same time,the protein expressionsof PCNA,TLR4,MyD88and NF-κB were decteted by Western blotting. Each set ofexperimental data used two-factor analysis of variance in statistics.In vitro: Taken venous blood vessels of mature AVF and cultured vascularsmooth muscle cells (VSMCs) by using tissue adherent method. VSMCs culturedsuccessfully were used for experiments. Observed the changes of PCNA,TLR4mRNA and protein expression levels at various concentration FBS in culture.Selected concentration of Fetal bovine serum (20%FBS),when TLR4expressedhighest.Then VSMCs were cultured with different concentrations of chitosan（0、10、100、500、1000、2000ug/ml）for48h. The mRNA expression of TLR4and PCNAwas studied by Real-time PCR, while the protein expression of PCNA,TLR4,MyD88 and NF-κB was studied by Western blotting. The cell immunofluorescence detectedTLR4and NF/κB protein expression and localization. Selected concentration ofchitosan (1000ug/ml),when TLR4expression was significantly altered by chitosantreating. And given different culture time (0,12,24,48h) to intervene. The mRNAexpression of TLR4and PCNA was studied by Real-time PCR, while the proteinexpression of PCNA,TLR4,MyD88and NF-κB was studied by Western blotting. Ineach group, data was analysed by one-way analysis of variance for comparison.Results:1．After4weeks rabbits intimal was thickening.In intimal hyperplasia a-SMAwas staining, and then proliferation of vascular smooth muscle cell was significant.As time increasing, more intimal hyperplasia shown obviously, the expression ofa-SMA significantly increased. Compared with model group, chitosan groupsignificantly reduced the degree of intimal hyperplasia, the level of a-SMA wassignificantly decreased,vascular smooth muscle cell proliferation was alsoextraordinarily decreased.2. Compared with normal control group, expression levels of PCNA, TLR4,MyD88and NF-κB increased with time. Chitosan groups were higher, butsignificantly lower than model groups.3．In vitro, high concentration FBS induced the higher expression of TLR4inVSMCs.In addition, the expression of TLR4was concentration dependent in adefinitive range. FBS-induced protein expression of PCNA,TLR4,MyD88andNF-κB were reduced by chitosan. They were dependent on concentration and time.Conclusion:1．Chitosan can inhibit the proliferation of rabbit VSMCs.It is that chitosancould downregulate TLR4-mediated signaling pathway,reducing the possibility ofintimal hyperplasia of rabbit AVF venous blood vessels.2．High concentration FBS induced VSMCs proliferation.3．Chitosan can be concentration-dependent and time-dependent inhibition ofVSMCs proliferation in high concentrations of FBS environment. The mechanismmay be related to activation of TLR4, reducing expression of downstream factorsMyD88and NF-κB. |
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