| Background and purposes:The abnormal increase and aggregation of?-synuclein??syn?is closely related to the pathogenesis and progression of Parkinson's disease?PD?.It can cause autophagic dysfunction,oxidative stress,endoplasmic reticulum stress and Ca2+concentration imbalance,and other dysfunction in dopaminergic neurons?DNs?.External?syn can also activate microglia,which can lead to autoimmune injury via inducing the release of pro-inflammatory cytokines.Therefore,how to promote the degradation of?syn in susbstantia nigra and inhibit the activation of microglia have gradually become new targets of PD therapy.Triptolide?T10?is an active ingredient extracted from Tripterygium wilfordii.It possesses anti-inflammatory,anti-tumor and immunomodulatory functions and is widely used in clinical treatment.Recent studies have shown that T10 can enhance the degradation of abnormal intracellular protein via inducing autophagy,but whether it can also effectively promote the degradation of?syn in DNs remains unclear.However,it has obvious hepatorenal toxicity and low blood-brain-barrier?BBB?pass rate,thus is limited in the treatment of intracranial diseases.Microbubbles?MBs?combined with focused ultrasound?FUS?is a new noninvasive BBB opening method,which can be used for intracranial targeted drug delivery.The purpose of this study was to explore the novel delivery method of T10 as well as the underlying mechanism in the treatment of PD.Methods:1.After mixing 14 mg DPPC and DSPE-PEG2000 at 5:2 molar ratio,adding 0.5 mg T10 and dissolving in chloroform,Triptolide-loaded microbubbles?T10-MBs?were prepared by film hydration method.The morphology of the microbubbles was observed by inverted fluorescence microscopy.The particle size distribution and zeta potential of the microbubbles were analyzed by particle size/potential analyzer.The drug loading rate of MBs was determined by high performance liquid chromatography-mass spectrometry?HPLC-MS?.2.The wild type?WT?and A53T mutations of?syn overexpressed adeno-associated virus?AAV?were constructed respectively.Male C57BL/6 mice,aged 6-8 weeks,were injected with 0.3?l virus at the stereotactic site of the right SN,and then fed for 4 months to construct the model of PD mice.3.Immediately after anaesthetizing and fixing the model mice and injecting T10-MBs via tail vein?containing about 4?g T10?,the right SN was irradiated by ultrasound using self-built low-frequency FUS equipment?probe frequency 620 kHz,curvature radius 80mm,pulse duration 10 ms,pulse frequency 1 Hz,total duration 60 s,negative pressure 0.45MPa?.The control group was administered by intraperitoneal injection directly twice a week for 3 weeks totally.The drug content in liver and brain of mice was determined by HPLC-MS to analyze the effect of different drug administration methods on drug distribution in vivo.Apoptotic cells were detected by TUNEL staining and the levels of alanine aminotransferase and glutamic oxaloacetate aminotransferase in liver were detected by biochemical analyzer to evaluate the hepatotoxicity of drugs.4.Western bloting was used to determine the expression of?syn monomers as well as high molecular weight aggregates of which,and dopamine transporters in substantia nigra.Immunofluorescence was used to detect the number of DNs in SN region.Enzyme-linked immunosorbent assay?ELISA?was used to elevate the content of dopamine?DA?in SN region.Thioflavine S?ThS?staining was used to detect the amyloid precipitation in SN.Rotarod test was used to elevate the motor balance and coordinatio.Western bloting was used to determine the expression of LC3II/LC3I,p62,ubiquitinated protein and Parkin.ELISA was used to determine Uch-L1 expression,and scanning electron microscopy was used to determine the number of autophagic bodies in DNs.5.Monomers,oligomers and pre-formed fibrils?PFFs?were prepared.After stimulating primary microglia with different forms of?syn at the same amount,inhibitors of TLR1/2,TLR4 and TLR9 were then added to the medium.The production of pro-inflammatory cytokines was determined by ELISA and the activity of NF-?B was determined by Western bloting.6.After co-treatment with PFFs and T10,SHIP1 inhibitor was added as the experimental group and PBS was added as the control group.The production of pro-inflammatory cytokines and NF-?B activity were compared by using ELISA and Western blot respectively.Results:1.The diameter of T10-MBs is 1724.0±48.8 nm,the dispersion coefficient is 0.149±0.006,the zeta potential is 16.98±2.4 mV,the drug loading rate is 83.6%.The optical microscope shows that MBs in solution are spherical-like in shape and uniform in size.2.In the control group,6 minutes after intraperitoneal administration,the peak concentration in liver reached 298.6±5.7 ng/g and in brain tissue 103.5±3.5 ng/g followed by gradual decline of drug concentration.In the experimental group,the peak concentration in brain tissue of mice appeared at the 3rd minute after administration,and the peak drug concentration was 467.6+8.6 ng/g,higher than that in the intraperitoneal injection group?P<0.05?,while the peak drug concentration in liver appeared at the 6th minute,and the peak drug concentration was 78.1+4.8 ng/g,lower than that in the intraperitoneal injection group?P<0.05?.TUNEL staining showed that intraperitoneal administration could induce hepatocyte apoptosis,while the number of apoptotic hepatocytes in the experimental group decreased significantly?P<0.05?.However,there were no obvious apopototic cells in brain tissue in the two group.Biochemical analysis showed that the levels of glutamic-oxaloacetic transaminase and alanine transaminase in the liver of mice in the control group increased significantly,suggesting liver injury,while the levels of which in the experimental group were significantly lower than those in the control group?P<0.05?.3.Compared with model group,the expression of?syn monomer and its high molecular weight aggregation in A53T/WT experimental group decreased significantly after treatment?P<0.05?,and the content of DA and dopamine transporter returned to normal level?P<0.05?.In the behavioral test,the falling time of mice in the experimental group was 32.6±6.3 s and 31.1±2.6 s respectively,while that of the blank control group was 7.8±1.9 s and 10.6±2.5 s respectively.There was significant difference between the two groups?P<0.05?.The latency time of mice in the intraperitoneal injection group was6.5±2.6 s and 13.1±2.2 s,respectively.There was no difference between the control group and the experimental group?P>0.05?.4.After treatment,the ratio of LC3II/LC3I increased significantly in the experimental group,and the expression of p62 decreased comparing with the blank control group?P<0.05?;the expression of Uch-L1,Parkin,ubiquitinated protein had no significant changes?P>0.05?.Meanwhile,results of scanning electron microscopy showed that the number of autophagic bodies in DNs in the experimental group was significantly higher than that in the control group?P<0.05?.5.PFFs have the strongest immunogenicity,followed by monomers and oligomers.Treatment with inhibitors of TLR1/2 and TLR4 abolished the PFFs-induced elevation of tumor necrosis factor alpha?TNF??and interleukin-1 beta?IL-??induced.6.T10 can inhibit the increase of NF-?B activity and the release of pro-inflammatory cytokines induced by PFFs,while SHIP1 inhibitor can eliminate the inhibitory effect of T10on the activity of NF-?B and the release of pro-inflammatory cytokines.Conclusions:T10-MBs-FUS treatment can significantly alleviate the motor dysfunction of PD model mice,but the same dose of drugs directly intraperitoneal injection has no obvious effect.According to the results of pharmacokinetics,T10 metabolizes rapidly in mice,and intraperitoneal injection can not fulfill effective therapeutic concentration of intracranial drugs.T10-MBs-FUS not only facilitates T10 delivery across the BBB,but also significantly reduces liver drug concentration,thus the hepatotoxicity.T10 promotes the degradation of?syn by inducing autophagy rather than activating proteasome.In addition,PFFs,an aggregate of?syn,activate microglia mainly through TLR1/2 and TLR4.T10 can inhibit PFFs-induced microglia activation through SHIP1/NF-?B signaling pathway,thus exerting neuroprotective effect. |
PDF Full Text Request