| Part Ⅰ:M-NPs preparation and characterizationObjective:To synthesize M-NPs with the function of macrophage cell membrane and prepare for the follow-up experiments.Methods:At first,the membrane from RAW264.7 mouse macrophages were obtained by gradient centrifugation after physical fragmentation,and the extracted cell membrane were observed under transmission electron microscope.Then,follow-up experiments were conducted to prove whether the extraction of macrophage cell membrane was successful.It is known that F4/80,TLR4,CD206 and CD11c are all surface markers of the outer membrane of macrophages,and LaminA/C is a nuclear marker.Follow-up experiments mainly included:(1)Western blot was used to detect the protein expression of f4/80,TLR4,CD206,CD11c and LaminA/C in Hom(homogenat),Mem(membrane),Nuc(nucleus),so as to determine whether the cell membrane extraction was successful.(2)PLGA nanoparticles were prepared by an improved double emulsification volatilization method;(3)The extracted macrophage cell membrane were mixed with PLGA nanoparticles in a certain proportion,and M-NPs were synthesized by repeated extrusion through the liposome extruder.(4)The morphology and diameter of macrophage cell membrane,PLGA NPs and M-NPs were detected by transmission electron microscopy.(5)The protein expressions of F4/80,TLR4,CD206,CD11c and LaminA/C in M-NPs were detected by Western blot.Results:(1)By means of gradient centrifugation after the macrophages were physically broken up,the cell membrane with high purity can be obtained.(2)Under electron microscopy,every synthesized PLGA nanoparticle was observed as spherical particle with a diameter of 171.4±17.3 nm.(3)Under electron microscopy,every synthesized M-NPs was composed of PLGA NPs core and outer membrane with a diameter of 214.1±18.6 nm.(4)Western blot analysis showed that F4/80,TLR4,CD206,CD11c and LaminA/C proteins were expressed in the homogenate.(5)F4/80,TLR4,CD206 and CD11c were expressed in the cell membrane,and LaminA/C was not expressed,which indicated high purity of the extracted cell membrane.(6)LaminA/C was only expressed in the nucleus,and other membrane surface marker proteins were not expressed.(7)M-NPs expressed F4/80,TLR4,CD206 and CD11c,but not LaminA/C.Conclusions:This part confirms that PLGA nanoparticles can be coated by macrophage cell membrane to form M-NPs.Part Ⅱ:M-NPs alleviate LPS-mediated macrophages activation.Objective:To explore the effect of M-NPs on LPS-mediated macrophage activation and the molecular mechanism in vitro.Methods:Extracting macrophage cell membrane,preparing PLGA nanoparticles,synthesizing and identifying M-NPs were according to the first part.Then RAW264.7macrophages in good growth state were divided into 6 groups.Each group was treated with 150 ng/ml LPS for 24 hours,and then were treated with M-NPs with concentrations of 0,0.5,1.0,2.0,3.0 and 5.0 mg/ml for 24 hours.Total proteins of each group were extracted,and the protein expressions of MyD88,IRAK1 and p-p65 were detected by Western blot.The residual LPS in the supernatant and the contents of TNF-α,IL-6 and IL-10 in the supernatant were detected by ELISA.Cell counting kit-8(CCK-8)detected the effect of different concentrations of NPs and M-NPs(0,0.5,1.0,2.0,3.0,and 5.0 mg/ml)on RAW264.7 marcophages growth activity to investigate whether M-NPs were cytotoxic to them.TLR4 receptor on RAW264.7 cell membrane was silenced or overexpressed byplasmid transfection.The transfection rate of plasmid was observed under fluorescence microscope 72 hours after plasmid transfection.The protein and nucleic acid expressions of TLR4 were detected by cell immunofluorescence assay,Western blot and RT-qPCR respectively.After successful plasmid transfection,the cell membranes of normal macrophages,silenced TLR4 macrophages and overexpressed TLR4macrophages were extracted to prepare M-NPs,silenced TLR4 M-NPs(TLR4~-/M-NPs)and overexpressed TLR4 M-NPs(TLR4~+/M-NPs),then RAW264.7 macrophages in good growth state were divided into 6 groups:Control group,M-NPs group,LPS group,LPS+M-NPs group,LPS+TLR4~+/M-NPs group,LPS+TLR4~-/M-NPs group.The control group did not get any treatment.In M-NPs group,the macrophages were treated with 1 mg/ml M-NPs for 24 h.In LPS group,the macrophages were treated with 150 ng/mL LPS for 24 hours.In LPS+M-NPs group,the macrophages were treated with 150 ng/mL LPS for 24 hours and then treated with 1 mg/mL M-NPs for 24 hours.In LPS+TLR4~+/M-NPs group,the macrophages were treated with 150ng/mL LPS for 24 hours and then treated with 1 mg/mL TLR4~+/M-NPs for 24 hours.In LPS+TLR4~-/M-NPs group,the macrophages were treated with 150 ng/mL LPS for24 hours and treated with 1 mg/mL TLR4~-/M-NPs for 24 hours.Then the total proteins of each group were extracted and the supernatant were collected for subsequent experiments:(1)The protein expressions of MyD88,IRAK1,p-p65 and p65 were detected by Western blot;(2)The residual LPS in supernatant was detected by ELISA.In order to detect the correlation between different concentrations of TLR4~+/M-NPs and M-NPs with supernatant residual LPS,RAW264.7 macrophages were treated with150 ng/mL LPS for 24 hours and then treated with different concentrations of TLR4~+/M-NPs or M-NPs for 24 hours.Results:(1)With using of plasmid transfection to silence or overexpresse the TLR4 receptor on RAW264.7 macrophages cell membrane,the transfection efficiency of plasmid was high and the silencing or overexpression of TLR4 was obvious.(2)M-NPs can effectively inhibit the protein expression of inflammatory signaling factors,such as MyD88,IRAK1 and p-p65.And the inhibitory effect was more obvious with the increase of concentration of M-NPs.(3)As the concentration of M-NPs increased,the content of residual LPS in the supernatant was lower,and when the M-NPs concentration was 2 mg/ml,the residual LPS in the supernatant was close to the lowest level.(4)With the increase of concentration of NPs or M-NPs,the inhibitory effect on cell growth became more obvious.When the concentration of NPs or M-NPs exceeded3 mg/ml,the inhibitory effect on cell growth was significant(P<0.05).(5)Compared with the LPS group,the protein expression levels of MyD88,IRAK1 and p-p65 in the M-NPs group were decreased,which suggested that M-NPs inhibited the activation of LPS mediated inflammatory signaling pathway in macrophages(P<0.05).(6)Compared with the M-NPs group,the protein expression levels of MyD88,IRAK1 and p-p65 in the TLR4~+/M-NPs group were lower,which declared that TLR4~+/M-NPs could inhibit the activation of LPS mediated inflammatory pathways more effectively(P<0.05).(7)After the silencing of TLR4,the inhibition of M-NPs on LPS mediated inflammation was reduced or disappeared.(8)When the concentration of TLR4~+/M-NPs or M-NPs was less than 3 mg/ml,the required concentration of TLR4~+/M-NPs was significantly lower than that of M-NPs to neutralize the same amount of LPS,and the difference was statistically significant(P<0.05).Conclusions:(1)M-NPs can neutralize LPS and reduce the binding of LPS to TLR4,then inhibit the inflammatorysignaling pathway of TLR4/MyD88/IRAK1/p-p65 and the release of TNF-αand IL-6 inflammatory factors.At last,M-NPs reduce the inflammatory response.And overuse of M-NPs was cytotoxic to macrophages.(2)TLR4~+/M-NPs was synthesized by over-expression of macrophage membrane TLR4 in vitro.When neutralizing the same dose of LPS,the dosage of nanomaterials could be effectively reduced,and the potential toxicity caused by excessive use of nanomaterials could be more effectively reduced.Part Ⅲ:M-NPs alleviate hepatic I/RI in liver transplantation.Objective:To investigate the effect of M-NPs on ischemia/reperfusion injury after liver transplantation in rats and the molecular mechanism.Methods:In order to study the distribution of M-NPs in rats,15 mg/kg NPs or M-NPs with red fluorescent probe were injected into SD rats by tail vein respectively,and the distribution of nanoparticles in rats was detected after 24 hours.The liver transplantation model of SD rats was established by double-sleeve method.30 SD rats were randomly divided into six groups,with five rats in each group,respectively as Blank control group(Blank),Liver transplantation group(Con),Liver transplantation+NPs group(NPs),Liver transplantation+M-NPs group(M-NP),Liver transplantation+TLR4~+/M-NPs group(TLR4~+/M-NPs),Liver transplantation+TLR4~-/M-NPs group(TLR4~-/M-NPs).Rats in the Blank group did not receive any treatment.The Con group only underwent liver transplantation.In other groups,20mg/kg of corresponding nanomaterials were injected through the tail vein after successful liver transplantation.4 days later,peripheral blood of each group was collected and the liver,heart,spleen,lung and kidney were removed for the following study:(1)HE staining was used to detect the pathological changes of heart,liver,spleen,lung and kidney.(2)Serum levels of ALT and AST were tested.(3)The level of serum LPS was detected by ELISA.Then different concentrations of M-NPs or TLR4~+/M-NPs were injected into the liver transplantation rats to test the LPS content in the serum.The effects of different concentrations of NPs or M-NPs on the survival of liver transplantation rats were further studied.After successful liver transplantation,60 SD rats were randomly divided into two groups with 30 rats in each group.Each large group was randomly divided into six small groups,with 5 rats in each small group.Different concentrations of NPs(0,50,100,150,200,250 mg/kg)or M-NPs(0,50,100,150,200,250 mg/kg)were injected into the rats by tail vein.Then the rats were observed continuously for 6 days,and the survival of the rats was counted.By the same way,30 SD rats were randomly divided into 6 small groups with five in each group,which were Blank control group(Blank),Liver transplantation group(Con),Liver transplantation+NPs group(NPs),Liver transplantation+M-NPs group(M-NPs),Liver transplantation+TLR4~+/M-NPs group(TLR4~+/M-NPs),Liver transplantation+TLR4~-/M-NPs group(TLR4~-/M-NPs).The KCs of the liver of the above 6 groups of rats were extracted for follow-up experiments:(1)The protein expressions of MyD88,IRAK1,p-p65 and p65 in KCs were detected by Western blot.(2)The nucleic acid levels of the pro-inflammatory factors TNF-αand IL-6 in KCs were detected by RT-qPCR.(3)The serum levels of TNF-αand IL-6 were detected by ELISA.Results:(1)NPs or M-NPs mainly expressed in the liver of rat,followed by the spleen,and was very few in other organs,such as heart,kidney and lung.(2)Compared with the Blank group,liver transplantation(Con group)significantly aggravated the pathological damage to rat heart,liver,spleen,lung and kidney,which mainly was reflected in the normal structure and texture changes of organs and the infiltration of a large number of inflammatory cells.(3)After liver transplantation,the injection of NPs(NPs group)had no significant effect on the injury of various organs caused by liver transplantation.(4)Compared with those in the Blank group,the pathological changes of heart,liver,spleen,lung and kidney in the M-NPs group were significantly reduced,and the protective effect of TLR4~+/M-NPs on all organs after liver transplantation was significantly higher than that in the M-NPs group.(5)The changes of serum ALT and AST were consistent with the results of HE staining.M-NPs could improve the abnormal liver function of rats caused by liver transplantation,while the improvement effect of TLR4~+/M-NPs on the liver function of rats was significantly better than that of M-NPs group.TLR4~-/M-NPs had no significant protective effect on the liver function of rats.(6)The change of serum level of LPS was also consistent with the result of HE staining,that is,M-NPs could significantly reduce the serum level of LPS in liver transplantation rats,and the reduction effect of TLR4~+/M-NPs was more significant than that of M-NPs,and the difference was statistically significant(P<0.05).(7)The reduction of serum LPS to the lowest level in liver transplantation rats requires about 30 mg/kg M-NPs or about 20 mg/kg TLR4~+/M-NPs.,which did not reach the safety threshold(100 mg/kg),and the amount of TLR4~+/M-NPs needed to reduce serum LPS to the same level was significantly lower than that of M-NPs(P<0.05).(8)Compared with the liver transplantation group,the protein expressions of MyD88,IRAK1 and p-p65 in KCs of the M-NPs group were significantly reduced(P<0.05),which suggested that M-NPs could effectively inhibit the activation of KCs in liver transplantation rats.(9)Compared with M-NPs group,the protein expressions of MyD88,IRAK1 and p-p65 in KCs of TLR4~+/M-NPs group were significantly reduced(P<0.05),which indicated that TLR4~+/M-NPs was more effective than M-NPs in inhibiting the activation of KCs in liver transplantation rats.The nucleic acid levels of TNF-αand IL-6 in KCs and the serum levels of TNF-αand IL-6 were similar to the results of Western blot,with statistically significant differences(P<0.05).Conclusions:(1)M-NPs was mainly distributed in the liver of rats after injection.(2)M-NPs alleviate hepatic ischemia reperfusion injury of rats who had liver transplantation by neutralizing endotoxin,and with the overexpression of TLR4,TLR4~+/M-NPs can more effectively alleviate ischemia-reperfusion injury.(3)M-NPs can inhibit the expression and secretion of inflammatory factors of KCs,and inhibit the secretion of anti-inflammatory factors,thus reduce ischemia reperfusion injury after liver transplantation.And the anti-inflammatory effect of TLR4~+/M-NPs is more obvious than that of M-NPs. |
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