| Background and objectivePneumocystis organisms are able to cause pneumonia inimmunocompromised animals and patients. Most human PCP cases areAIDS-related, but PCP also occurs in other severely immunocompromisedpatient populations, including those with anti-cancer or immunosuppressivetherapy. Due to the increasing of Pneumocystis infected patients, PCP hasattracted more and more attention.So far, the most effective treatment for PCP is chemical therapy,including Trimethoprim-Sulfamethoxazole (Tmp-Smz), pentamidine,dapsone and atovaquone. et al. However, negative side effects, limitedefficacies and drug resistance are associated with these drugs. Because ofemergence of side effects and drug resistance in PCP’s therapy, adevelopment of new drugs for PCP is urgent. Chitosan is easily obtained by deacetylation of chitin, and has shownmore advantages in applications for its, nontoxic, biocompatible andbiodegradable properites. Chitosan has been reported to show a widespectrum of inhibition against microorganisms such as bacteria and fungi.Zymosan from Saccharomyces cerevisiae can be used as activator forDectin-1receptor, which plays a role in immune regulation. Studies havefound that Dectin-1receptor resistant to Pneumocystis infection, PCPpatients with Dectin-1receptor expression down-regulate, zymosan canup-regulate the expression of Dectin-1receptor in order to enhance the antiPc infection effect. To our knowledge, however, no research has beenconducted exploring the efficacy of chitosan and zymosan for the treatmentof PCP so far. The study of anti-PCP effect is base the chitosan antibacterialaction, zymosan up-regulate Dectin-1receptors, and chitosan-zymosanmicrosphere sustained release. The study will provide a theoretical basis forthe chitosan and zymosan on PCP.The purpose of this study is to investigate the therapeutic effect ofchitosan and chitosan-zymosan microsphere on PCP, and to explore itsanti-PCP mechanism.Methods1. Immunosuppressed rat PCP model and experimental groups fortherapy with chitosanRats were immunosuppressed by intramuscular injections of dexamethasone sodium phosphate once daily for2days/week. The ratsconfirmed the presence of P. carinii infection were randomly divided into7groups: immune normal group,0.5%chitosan-immune normal group, PCPmodel group, Tmp-Smz group (Tmp0.0215mg/g and Smz0.11mg/g bodyweight),0.2%acetic acid group,0.1%chitosan group and0.5%chitosangroup.2. Investigate the effect of0.5%chitosan on PCP rats by weight gain,lung weight, lung weight/body weight (LW/BW) ratio and survivalpercentagesThe model rats were weighed at pre-and post-therapy. The rats weresacrificed, and lungs were aseptically excised and weighed. By the test rats’weight gains, lung weights, LW/BW ratios and survival percentages of rats,the evaluation of the anti-PCP therapeutic effects was conducted.3. Study the effect of0.5%chitosan on PCP rats’ lung tissuesThe rat lung tissues were stained with hematoxylin and eosin (HE) tovisualize lung histological changes and inflammatory infiltration by lightmicroscopy examination.4. Observe ultrastructural changes of P. carinii by scanning electronmicroscope (SEM) and transmission electron microscope (TEM)The rat lung tissues were prepared as regular methods for electronmicroscopic examinations. The ultrastructural damages of P. carinii wereobserved by SEM and TEM. 5. Assess HSP70mRNA expression of P. carinii by quantitative real-timePCR analysisTotal RNA was extracted fung tissue, rat lung tissue P. carinii HSP70mRNA expression levels were measured by real-time PCR.6. Measure rat serum levels of IL-10, IFN-γ and TNF-α by ELISAThe rat serum of abdominal aorta blood was collected. Effects ofdrugs for rat serum IL-10, IFN-γ and TNF-α concentration were measuredby ELISA.7. Investigate numbers of rat CD4+T, CD8+T lymphocytes by flowcytometryThe rat abdominal aorta blood was collected. Effects of drugs for ratCD4+T, CD8+T lymphocytes numbers were determined by flow cytometry.8. Immunosuppressed rat PCP model and experimental groups fortherapy with chitosan-zymosan microsphereRats were immunosuppressed by intramuscular injections ofdexamethasone sodium phosphate once daily for2days/week. The ratsconfirmed the presence of P. carinii infection were randomly divided into7groups: immune normal group, PCP model group,0.5%chitosan group,0.5%chitosan+0.9%NaCl group,0.5%chitosan+0.5%zymosangroup,0.5%chitosan+1%zymosan group,0.8%chitosan-zymosanmicrosphere group.9. Investigate the effect of0.8%chitosan-zymosan microsphere on PCP rats by weight gain, lung weight, LW/BW ratio and survival percentagesThe model rats were weighed at pre-and post-therapy. The rats weresacrificed, and lungs were aseptically excised and weighed. By the test rats’weight gains, lung weights, LW/BW ratios and survival percentages of rats,the evaluation of the anti-PCP therapeutic effects was conducted.10. Study the effect of0.8%chitosan-zymosan microsphere on PCPrats’ lung tissuesThe rat lung tissues were stained with HE to visualize histologicalchanges and inflammatory infiltration by light microscopy examination.11. Observe ultrastructural changes of P. carinii by TEMThe rat lung tissues were prepared as regular methods for electronmicroscopic examinations. The ultrastructural damages of P. carinii wereobserved by TEM.12. Assess Dectin-1and TLR-4mRNA expression by quantitativereal-time PCR analysisTotal RNA was extracted from lung tissue, effects of drugs for rat lungtissue Dectin-1and TLR-4mRNA expression levels were measured byreal-time PCR.13. Assess HSP70mRNA expression of P. carinii by quantitative real-timePCR analysisTotal RNA was extracted from lung tissue, rat lung tissue P. cariniiHSP70mRNA expression levels were measured by real-time PCR. 14. Measure rat serum levels of cytokine by ELISAThe rat serum of abdominal aorta blood was collected. Effects ofdrugs for rat serum IL-1β、IL-4、IL-10、IFN-γ、MCP-1、IL-1α、IL-2、IL-6and TNF-αconcentration were measured by ELISA.Results1. The effect of0.5%chitosan on PCP rats weight gain, lung weight,LW/BW ratio and survival percentagesThe weight gain of rats in the0.5%chitosan group was significantlyincreased compared to the PCP model,0.2%acetic acid and0.1%chitosan groups, respectively. The lung weight and LW/BW ratio wassignificantly reduced in the0.5%chitosan group, compared with the PCPmodel,0.2%acetic acid and0.1%chitosan groups, respectively. Theresults indicate that rats treated with0.5%chitosan had an improvedchance of survival compared with the PCP model and0.2%acetic acidgroups.2. The effect of0.5%chitosan on PCP rats’ lung tissuesThe alveolar spaces foamy material, the numbers of inflammatory celland alveolar macrophages in the0.5%chitosan were significantlydecreased compared to the PCP model,0.2%acetic acid and0.1%chitosan groups, respectively.3. The effect of0.5%chitosan on P. carinii ultrastructuralAfter0.5%chitosan therapy, the sections revealed that P. carinii nuclear structure atrophy, and incomplete wall structures under electronmicroscopy.4. The effect of0.5%chitosan on rat lung tissue P. carinii HSP70mRNAexpressionTherapeutic treatment with0.5%chitosan significantly reducedHSP70mRNA expression, compared with the PCP model,0.2%aceticacid and0.1%chitosan groups, respectively.5. The effect of0.5%chitosan on rat serum levels of IL-10, IFN-γ andTNF-αThe IL-10and IFN-γ secretion was significantly higher in the0.5%chitosan group, compared with the PCP model,0.2%acetic acid and0.1%chitosan groups, respectively. TNF-α secretion was significantly lower inthe0.5%chitosan group, compared with the PCP model,0.2%acetic acidand0.1%chitosan groups, respectively.6. The effect of0.5%chitosan on rat abdominal aorta blood CD4+T,CD8+T lymphocytes numbersThe CD4+T lymphocyte numbers significantly increased in the0.5%chitosan group, compared with the PCP model,0.2%acetic acid and0.1%chitosan groups, respectively. The CD8+T lymphocyte numbers weresignificantly reduced in the0.5%chitosan group, compared with the PCPmodel and0.2%acetic acid groups, respectively.7. The effect of0.8%chitosan-zymosan microsphere on PCP rats weight gain, lung weight, LW/BW ratio and survival percentagesThe weight gain of rats in the0.8%chitosan-zymosan microspheregroup was significantly increased compared to the PCP model,0.5%chitosan and0.5%chitosan+0.5%zymosan groups, respectively. Thelung weight and LW/BW ratio was significantly reduced in the0.8%chitosan-zymosan microsphere group, compared with the PCP model,0.5%chitosan and0.5%chitosan+0.5%zymosan groups, respectively.The results indicate that rats treated with0.8%chitosan-zymosanmicrosphere had an improved chance of survival compared with the PCPmodel group.8. The effect of0.8%chitosan-zymosan microsphere on PCP rats’ lungtissuesThe numbers of inflammatory cell in the0.8%chitosan-zymosanmicrosphere were significantly decreased compared to the PCP model,0.5%chitosan and0.5%chitosan+0.5%zymosan groups, respectively.9. The effect of0.8%chitosan-zymosan microsphere on rat lung tissueDectin-1and TLR-4mRNA expressionTherapeutic treatment with0.8%chitosan-zymosan microspheresignificantly increased Dectin-1and reduced TLR-4mRNA expression,compared with the PCP model,0.5%chitosan and0.5%chitosan+0.5%zymosan groups, respectively.10. The effect of0.8%chitosan-zymosan microsphere on rat lung tissue P. carinii HSP70mRNA expressionTherapeutic treatment with0.8%chitosan-zymosan microspheresignificantly reduced HSP70mRNA expression, compared with the PCPmodel,0.5%chitosan and0.5%chitosan+0.5%zymosan groups,respectively.11. The effect of0.8%chitosan-zymosan microsphere on rat serum levelsof cytokineThe IL-1β, IL-4, IL-10and IFN-γ secretion was significantly higher inthe0.8%chitosan-zymosan microsphere group, compared with the PCPmodel,0.5%chitosan and0.5%chitosan+0.5%zymosan groups,respectively. IL-1α, IL-6and TNF-α secretion was significantly lower inthe0.8%chitosan-zymosan microsphere group, compared with the PCPmodel,0.5%chitosan and0.5%chitosan+0.5%zymosan groups,respectively. The MCP-1secretion was significantly higher and IL-2secretion was significantly lower in the0.8%chitosan-zymosanmicrosphere group, compared with the PCP model.Conclusion1. The0.5%chitosan had some apparent treatment effects on PCP byreducing P. carinii HSP70mRNA expression, the LW/BW ratio and lunginflammatory cell infiltration. Additionally, it increased the concentrationsof IFN-γ and IL-10as well as the CD4+T lymphocyte numbers, andreduced the CD8+T lymphocyte numbers and concentration of TNF-α in PCP model rats. Finally, chitosan was also found to cause seriousultrastructural damage to P. carinii.2. The0.8%chitosan-zymosan microsphere had some apparent treatmenteffects on PCP by reducing P. carinii HSP70mRNA, lung tissue TLR-4mRNA expression, the LW/BW ratio and lung inflammatory cellinfiltration. Additionally, it increased lung tissue Dectin-1mRNAexpression, the concentrations of IL-1β, IL-4, IL-10, IFN-γ and MCP-1,and reduced the concentration of IL-1α, IL-2, IL-6and TNF-α in PCPmodel rats. Finally,0.8%chitosan-zymosan microsphere was also found tocause serious ultrastructural damage to P. carinii. |
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