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OBJECTIVE: To synthesize low molecular weight chitosan-acetylcysteine (LMWC-NAC) conjugate and investigate its renal targeting profile and the rapeutic effects in model mice with acute kidney injury (AKI).METHODS: NAC was conjugated to LMWC by EDC/NHS reaction and the LMWC-NAC conjugate was identified by 1H-NMR. The cellular uptake of LMWC-NAC conjugate and megalin receptor involved in this process was investigated in vitro. In addition, the tissue distribution of ICG-labelled LMWC-NAC conjugate was investigated in nude mice. AKI were induced by LPS intraperitoneal injection (20 mg·kg-1).The parameters including Scr, BUN, inflammatory factors (TNF-α and IL-1β), and oxidative stress (MDA) were determined and renal histology was observed. RESULTS: LMWC-NAC conjugate was successfully synthesized by the amide interaction.The in vitrostudies demonstrated that the uptake of LMWC-NAC conjugate was mediated by the megalin receptor on HK-2 cells, and the tissue distribution experiment indicated that LMWC-NAC conjugate was mainly accumulated in the kidney.LMWC-NAC conjugate significantly suppressed Scr, BUN, inflammatory factors and oxidative stress (P<0.01) and improved kidney injury. CONCLUSION: LMWC-NAC conjugate showed good renal targeting profile and effect in recovering renal functions, which indicates the potential of LMWC-NAC conjugate as a safe and efficient drug delivery system for the treatment of AKI.
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OBJECTIVE: To prepare conjugate of low molecular weight chitosan(LMWC) with glycyrrhizin (GL) and investigate its cellular uptake by proximal tubular epithelial cells (HK-2). METHODS: Glycyrrhizin-LMWC (GL-LMWC) conjugate was synthesized by the reaction between the amino group of LMWC and the active ester group of GL. Drug release was studied in phosphate buffer (pH 4.0) containing papain. The uptake of the conjugate by HK-2 cells and its intracellular distribution was studied with laser confocal microscopy. RESULTS: The chemical structure of GL-LMWC conjugate was confirmed by IR and H-NMR. The GL grafting rate was determined to be 29.3%. The drug liberation from the conjugates was significantly accelerated in the presence of papain, which confirmed the capability of the conjugate to degrade in lysosomes. The conjugate could be internalized by HK-2 cells. CONCLUSION: The GL-LMWC conjugate with well-defined structure is successfully synthesized and exhibitts high cellular uptake efficiency. The conjugate has the potential to retaing GL in the kidney for a prolonged duration and to sustain its release locally for better efficacy.
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Objective To clarify the inhibitory effect of low molecular weight chitosan(LMCTS)on hepatic fibrosis induced by carbon tetrachloride (CCl4 )in the rats, and to investigate its effect on the expression of Toll-like receptor-4(TLR4 )and to lay the foundation for the development of the clinical candidate drug of liver fibrosis. Methods 72 male Wistar rats were randomly divided into blank control group, CCl4 group (model group), glycyrrhizinate (DG)group,50,100 and 150 mg·kg-1 LMCTS groups (low,middle and high doses of LMCTS groups).In addition to blank control group,the rats in the remaining groups were given 40% CCl4-vegetable oil (1.75 mL·kg-1 ),2 times per week for 8 weeks,by intraperitoneal injection to establish the model of rat hepatic fibrosis.And the rats in blank control group were injected with the same amount of 100% vegetable oil agent. From the ninth week, the rats in DG and LMCTS groups were given DG and LMCTS by intragastric administration, 1 time/week for 4 weeks. Then all rats were sacrificed, the activities of serum glutamic acid aminotransferase (ALT),aspartate aminotransferase (AST)and alkaline phosphatase (ALP)were detected with ELISA kit;the pathological changes in liver tissue were observed under light microscope, and the TLR4 expressions were detected by RT-PCR and Western blotting method. Results The serum ALT, AST and ALP activities in middle and high doses of LMCTS groups were lower than those in model group (P0.05).There were statistically significant differences in the serum ALT, AST and ALP activities between high dose of LMCTS group and middle dose of LMCTS group (P<0.05).There were obvious hepatocyte steatosis,inflammatory cell infiltration,collagen fiber hyperplasia and hepatic lobule damage in the rats in model group.However,all the changes in liver tissue of the rats in LMCTS group were significantly reduced, especially in high dose group. The results of RT-PCR and Western blotting method showed that the expression of TLR4 was declined in LMCTS groups compared with model group (P<0.05,P<0.01),especially in high dose of LMCTS group,and there were statistically significant differences between different doses of LMCTS groups (P<0.05).Conclusion High dose of LMCTS can decrease the serum transaminase activity of liver fibrosis rats and improve liver function,and this process may be related to declining the expression of TLR4 .