Experimental Study Of Gold Nanoclusters Inhibiting Inflammatory Response In Ischemic Stroke By Regulating Microglial Polarization

ObjectiveThe aim of this study is to construct gold nanoclusters(Au NCs),detect the therapeutic effect of Au NCs on ischemic stroke,and explore its mechanism of inhibiting inflammatory response by regulating microglial polarization.Methods1.The Au NCs were prepared by Na OH reduction system,and the morphological characteristics of the materials were observed by transmission electron microscope(TEM),UV-visible light spectrophotometer,dynamic light scattering particle size analysis(DLS),and fluorescence visible spectrophotometer.The biological safety of Au NCs was detected by HE staining.2.Animal experiment: healthy male SD rats were randomly divided into three groups: Sham operation group(Sham),Middle cerebral artery occlusion group(MCAO),Au NCs treatment group(MCAO+Au NCs).The rats in the treatment group were injected with Au NCs(10μL/1m L)through the tail vein 1h after infarction.The brain tissues were collected 24 h after infarction.The neurological function of rats was detected by m NSS neurobehavioral score and fatigue balance rotarod test.The cerebral infarction volume was detected by TTC staining,and the neuronal damage was detected by Nissl staining,HE staining,and Neu N fluorescence staining.The expression changes of IBA1,TNF-α,TGF-β1,p-JAK2,and p-STAT3 were detected by immunofluorescence staining.The protein levels of TNF-α,IL-1β,IL-6,TGF-β1,IL-10,ARG-1,pJAK2,JAK2,p-STAT3 and STAT3 were detected by Western blot.3.Cell experiment: microglial cells(Bv2)were cultured in vitro and induced by LPS to construct an in vitro inflammatory cell model.The experiment was randomly divided into three groups: Control,LPS and Au NCs(LPS+Au NCs).Follow-up experiments were conducted in LPS and Au NCs groups after incubation for 24 h.The concentration of Au NCs was determined to be 4μL/m L by CCK8 method.Immunofluorescence staining was used to detect the expression of tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1).q PCR was used to detect the m RNA expression of TNF-α,IL-6,IL-1β,and IL-10.The expressions of JAK2,STAT3,p-JAK2 and p-STAT3 were detected by immunofluorescence staining and Western blot.Results1.The 15 nm ultra-small Au NCs constructed in this study have uniform particle size,regular morphology,no obvious toxic side effects,and high biological safety.2.The results of animal experiments:(1)The results of m NSS neurobehavioral score and fatigue balance rotarod test showed that the neurological function of MCAO group was severely impaired,and Au NCs treatment group had a certain degree of improvement.(2)TTC staining results showed that Au NCs significantly reduced cerebral infarction area.(3)HE staining,Nissl staining,and Neu N fluorescent staining showed a large number of neuronal apoptosis in the cerebral cortex of MCAO group.(4)Western blot and immunofluorescence staining showed that compared with sham group,the expression of IBA1,TNF-α,IL-1β and IL-6 in MCAO group increased,while the expression of TGF-β1,IL-10 and ARG-1 decreased.The expressions of IBA1,TNF-α,IL-1β and IL-6 in Au NCs treatment group were significantly decreased,while the expressions of TGF-β1,IL-10 and ARG-1 were significantly increased.(5)The results of immunofluorescence staining and Western blot confirmed that the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 increased after cerebral ischemia,and Au NCs treatment inhibited the activation of JAK/STAT3 pathway.3.Results of cell experiments:(1)The optimal concentration of Au NCs was determined to be 4μL/m L by CCK8 method.(2)q PCR results showed that compared with the control group,the m RNA expression of TNF-α,IL-1β and IL-6 in the LPS group increased,and the m RNA expression of IL-10 decreased.Compared with LPS group,the expression of TNF-α,IL-1β and IL-6 m RNA in Au NCs group decreased significantly,and the expression of IL-10 m RNA increased.(3)The results of immunofluorescence staining showed that the expression of TNF-α increased and the expression of TGF-β1 decreased in the LPS group,and the expression of TNF-α decreased and the expression of TGF-β1 increased in the Au NCs treatment group.(4)The results of immunofluorescence staining showed that the expression of p-JAK2 and pSTAT3 in BV2 cells was increased after LPS stimulation,and the fluorescence density of p-JAK2 and p-STAT3 was decreased after Au NCs treatment.The results of Western blot showed that the ratio of p-JAK2/JAK2 and pSTAT3/STAT3 in Au NCs group was significantly lower than that in LPS group.ConclusionsThe Au NCs constructed in this study are small in size and can effectively inhibit inflammatory response,protect neurons,and treat ischemic stroke.The mechanism may be achieved by regulating the polarization of microglia through the JAK2/STAT3 pathway.