Experimental Study On Promoting Spinal Cord Injury Repair By Gold Nanoclusters Loaded With Berberine Through Mediating Macrophage/microglia Polarizatio

Objective:Gold nanoclusters(Au NCs)loaded with berberine(BRB)was designed and constructed to prepare BRB-Au NCs.Furthermore,in vitro and in vivo experiments were carried out to explore the molecular mechanism of BRB-Au NCs regulating inflammatory response and inhibiting neuronal apoptosis after spinal cord injury(SCI),providing new strategies and ideas for the treatment of SCI.It also provides a new way to improve the drug utilization rate by using Au NCs,and provides a theoretical basis for the clinical application of BRB in the treatment of SCI.Materials and methods:1.Part IThe preparation and characterization of BRB-loaded Au NCs.Au NCs were prepared by using bovine serum albumin(BSA)as reducing agent and protective agent.Au NCs loaded with BRB(BRB-Au NCs)were prepared by mixing Au NCs with BRB.The encapsulation efficiency(EE)and load capacity(LC)were measured and calculated by high performance liquid chromatography.The morphology and size of Au NCs were observed by transmission electron microscopy.The Zeta potentials of BRB,Au NCs and BRB-Au NCs at different p H(5.5,6.8 and 7.4)were measured by laser particle size analyzer.The optical properties of BRB,Au NCs and BRB-Au NCs were tested by ultraviolet-visible spectrophotometer and fluorescence spectrophotometer.The drug release characteristics of BRB-Au NCs were measured by high performance liquid chromatography.The SCI model was established by Allen method.The experimental animals were divided into two groups : BRB group and BRB-Au NCs group.BRB or BRB-Au NCs were injected into the tail vein.After 24 hours of injection,the spinal cord,heart,liver,spleen,lung and kidney were taken out.The in vivo distribution of BRB and BRB-Au NCs was measured by high performance liquid chromatography.2.Part IIRAW 264.7 cells were cultured in vitro.The effects of different concentrations(0,1,10,50,100 and 500 μM)of BRB and BRB-Au NCs on cell viability were determined by MTT assay.Lipopolysaccharide(LPS)was used to induce cellular inflammatory response.The cells were randomly divided into five groups: normal control group,LPS treatment group,LPS +Au NCs group,LPS + BRB group,LPS + BRB-Au NCs group.After 24 hours of LPS induction,50 μM BRB or 50 μM BRB-Au NCs were given respectively.The expression of IL-1β,IL-6 and TNF-α were detected by Western blot.SCI rat model was established by Allen method.Experimental animals were randomly divided into four groups: Sham group,SCI + saline group,SCI + BRB group,SCI + BRB-Au NCs group.After 3 days after SCI,the protein of spinal cord was extracted,and the expression of IL-1β,IL-6 and TNF-α were detected by Western blot.3.Part IIIVSC 4.1 cells were cultured in vitro.The effects of different concentrations(0,1,10,50,100 and 500 μM)of BRB and BRB-Au NCs on cell viability were determined by MTT assay.LPS induces cellular inflammatory response.The cells were randomly divided into five groups:control group,LPS treatment group,LPS + Au NCs group,LPS + BRB group,LPS +BRB-Au NCs group.After 24 hours of LPS induction,50 μM BRB or 50 μM BRB-Au NCs were given,and the expression of apoptosis-related proteins Cleaved Caspase-3,Bax and Bcl-2 was detected by Western blot.The expression of apoptotic protein Cleaved Caspase-3was detected by immunofluorescence staining.SCI rat model was established by Allen method.The experimental animals were randomly divided into four groups: Sham group,SCI+ saline group,SCI + BRB group,SCI + BRB-Au NCs group.The expression of apoptosis-related proteins Cleaved Caspase-3,Bax and Bcl-2 was detected by Western blot.BBB score(Basso,Beattie & Bresnahan locomotor rating scale)and inclined plane test were used to observe the recovery of motor function in rats 1,3,7,14,21 and 28 days after SCI.The histomorphological recovery of the injured area after 28 days of SCI was observed by HE staining.The expression of Nissl bodies in spinal anterior horn motor neurons was observed by Nissl staining at 28 days after SCI,and the number of Nissl bodies was calculated.The expression of apoptotic protein Cleaved Caspase-3 in spinal anterior horn motor neurons was observed by immunofluorescence staining at 3 days after SCI.4.Part IVRAW 264.7 cells were cultured in vitro.LPS induced cellular inflammatory response.The cells were randomly divided into five groups: control group,LPS treatment group,LPS +Au NCs group,LPS + BRB group,LPS + BRB-Au NCs group.After 24 hours of LPS induction,50μM BRB or 50μM BRB-Au NCs were given respectively.Western blot was used to detect the expression of NF-κB signaling pathway-related proteins t-NF-κB,p-NF-κB,IKKβ and NLRP3 inflammasome-related proteins NLRP3,ASC,Caspase 1,IL-1β,as well as the expression of M1 marker protein CD86 and M2 marker protein CD206.The expression of p-NF-κB,NLRP3,CD86 and CD206 in each group was detected by immunofluorescence staining.The rat SCI model was established by Allen method.The experimental animals were randomly divided into four groups: Sham group,SCI + saline group,SCI + BRB group,SCI +BRB-Au NCs group.After 3 days of SCI,the expression of NF-κB signaling pathway related proteins t-NF-κB,p-NF-κB,IKKβ and NLRP3 inflammasome related proteins NLRP3,ASC,Caspase 1,IL-1β were detected by Western blot.The expression of microglia marker protein CD11 b in the injured area was observed by immunofluorescence staining.The toxicity of BRB-Au NCs to various organs in rats injected with drugs was observed by HE staining.Results:Part I1.Transmission electron microscopy was used to observe that BSA-Au NCs was a small cluster with an average size of about 2 nm.At the same time,the measurement and statistical results of the dynamic size of Au NCs and BRB-Au NCs by Nano Measure 1.2 show that the dynamic size of Au NCs is 1.573± 0.57 nm,while the dynamic size of BRB-Au NCs is121.982± 0.913 nm.Obviously,the size increased significantly after adding BRB,indicating that there was a conjugated relationship between BRB and Au NCs aggregates,and the material was successfully synthesized.In addition,by observing BRB and BRB-Au NCs dissolved in BSA,it can be seen that BRB is not completely dissolved in BSA,and there are suspended particles.After standing for 1 hour,the particles settle into precipitates.The BRB modified by Au NCs was almost completely dissolved in BSA,and no precipitation was formed after standing for 1 hour,which indirectly indicated that Au NCs improved the solubility of BRB.2.The results of UV-Vis spectrophotometer and fluorescence spectrophotometer showed that BRB-Au NCs combined the optical properties of BRB and Au NCs.After the combination of Au NCs and BRB,the peak shifted significantly,indicating that Au NCs may interact with BRB and BRB was successfully loaded into Au NCs.3.Zeta potential results showed that unlike BRB and Au NCs with only one Zeta potential peak,BRB-Au NCs had multiple peaks,resulting in an average potential conversion between different peak positions.It is worth noting that the main Zeta potential peaks of BRB-Au NCs can be switched from negative charge to positive charge under different p H environments(i.e.,5.5,6.8 and 7.4),indicating that BRB-Au NCs exhibit zwitterionic drugs.The positive charge is conducive to the interaction between the drug and the negatively charged cells,while the negative charge can protect the cells from excessive damage.4.The measurement and calculation results of high performance liquid chromatography showed that the encapsulation efficiency of BRB-Au NCs was 64.10±1.69 %,and the drug loading rate was 8.28±0.28 %,indicating that the synthesized BRB-Au NCs had high drug utilization rate.At 24 h,the cumulative release of BRB-Au NCs reached 70.67 ± 3.05 %.At the same time,after SCI modeling in rats,BRB was more distributed in the SCI area of rats,which proved that BRB-Au NCs significantly improved the absorption rate of BRB.Part II1.RAW 264.7 cells were cultured in vitro.MTT results showed that when the concentration of BRB and BRB-Au NCs exceeded 100μM,the cell activity began to decrease,but the overall survival rate was still above 80%.When the concentration of BRB or BRB-Au NCs was low,the cell viability was almost 100%,and low concentrations of BRB or BRB-Au NCs were not toxic to RAW 264.7 cells.2.LPS was used to induce inflammation model.The results showed that BRB-Au NCs reduced the expression of inflammation-related proteins TNF-α,IL-1β and IL-6 in RAW264.7 cells,and the anti-inflammatory effect was more significant than that of BRB(P＜0.001).3.The results of in vivo experiments showed that BRB-Au NCs inhibited the expression of inflammation-related proteins TNF-α,IL-1β and IL-6 in the injured area at 3 days after SCI.Compared with BRB group,the expression levels of TNF-α,IL-1β and IL-6 in BRB-Au NCs group were decreased(P＜0.001),indicating that Au NCs improved the anti-inflammatory effect of BRB and improved the bioavailability of BRB.Part III1.VSC 4.1 cells were cultured in vitro.MTT results showed that when the concentration of BRB and BRB-Au NCs exceeded 100μM,the cell activity began to decrease,but the overall survival rate was still above 80%.When the concentration of BRB or BRB-Au NCs was low,the cell viability was almost 100%,and low concentrations of BRB or BRB-Au NCs were not toxic to VSC 4.1 cells.2.LPS induced inflammation model.Western Blot results showed that BRB-Au NCs significantly reduced the expression of apoptosis-related proteins Cleaved Caspase-3 and Bax in VSC 4.1 cells,and promoted the expression of anti-apoptotic protein Bcl-2,which was stronger than BRB(P＜0.001).The results of immunofluorescence staining were consistent with the results of Western Blot,with significant difference(P＜0.001).3.The results of in vivo experiments showed that from the 7th day,the motor function scores of BRB and BRB-Au NCs were significantly higher than those of the control group,and the increasing trend of BRB-Au NCs group was more obvious,indicating that BRB-Au NCs promoted the recovery of motor function after SCI.At the same time,the area of the injured area in the BRB-Au NCs group was significantly reduced,and the survival of motor neurons in the anterior horn of the spinal cord was improved.Compared with the BRB group,the results were significantly different(P＜0.001).4.Immunofluorescence results of spinal cord sections showed that BRB-Au NCs significantly inhibited the expression of apoptotic protein Cleaved Caspase-3 in injured spinal cord anterior horn motor neurons.At the same time,Western Blot results showed that BRB and BRB-Au NCs significantly inhibited the expression of apoptosis-related proteins Cleaved Caspase-3 and Bax after SCI in rats,while the anti-apoptotic protein Bcl-2 was activated and the ratio of Bax / Bcl-2 was increased.The decreasing trend of BRB-Au NCs group was more obvious than that of BRB group,and the results were significantly different(P＜0.001).This indicates that BRB-Au NCs can more effectively inhibit neuronal apoptosis after SCI than BRB.Part IV1.The expression of t-NF-κB,p-NF-κB and IKKβ protein in RAW 264.7 cells was detected by Weatern Blot.The results showed that BRB and BRB-Au NCs could effectively inhibit the phosphorylation of NF-κB.At the same time,the expression of NLRP3 inflammasome-related proteins NLRP3,ASC,Caspase 1 and IL-1β was also significantly reduced.Moreover,the decreasing trend caused by BRB-Au NCs was more obvious,indicating that BRB-Au NCs inhibited the activation of NLRP3 inflammasome through NF-κB signaling pathway and improved the therapeutic efficiency of BRB.2.Western blot and immunofluorescence showed that the expression of CD86(M1 signature protein)was increased in LPS-stimulated RAW 264.7 cells,while the expression of CD206(M2 signature protein)was decreased,indicating that the cells were polarized into M1 pro-inflammatory cells after treatment with LPS in vitro.On the other hand,the BRB group and BRB-Au NCs group enable the changing trend of the two markers more dramatically,while BRB-Au NCs most significantly increased CD86 while decreasing CD206.These results indicated that BRB-Au NCs inhibited the activation of NLRP3 inflammatorome through NF-κB signaling pathway,thereby reducing the polarization of macrophages towards M1 phenotype,increasing the polarization of M2 phenotype,and improving the therapeutic efficiency of BRB.3.BRB-Au NCs effectively reduced the phosphorylation of NF-κB and the expression of IKKβ after 3 days of SCI,inhibited the expression of NLRP3 inflammator-related protein,and improved the bioavailability of BRB in vivo.These results indicated that BRB-Au NCs inhibited the activation of NLRP3 inflammatories after SCI through NF-κB signaling pathway,and improved the bioavailability of BRB in vivo,which further increased the anti-inflammatory ability of BRB.4.CD11 b was used to label macrophages/microglia in spinal cord sections at 3 days after injury,and it was found that BRB-Au NCs effectively inhibited the activation of macrophages/microglia and reduced their polarization to M1 phenotype,showing a better effect than the BRB group.5.Various organs and tissues(heart,liver,spleen,lung and kidney)of rats injected with BRB-Au NCs were stained by HE staining,and it was found that there was no organ injury after injection of BRB-Au NCs compared with the normal control group,which indicated that BRB-Au NCs was safe in vivo.Conclusions:1.In this study,BRB-Au NCs were successfully prepared and the absorption rate of BRB was significantly improved.2.BRB-Au NCs significantly inhibited the expression of inflammatory factors after SCI,and showed more significant anti-inflammatory ability than BRB.3.BRB-Au NCs significantly inhibited nerve cell apoptosis and promoted motor function recovery after SCI.4.BRB-Au NCs inhibit the activation of inflammatory bodies through the NF-κB pathway,thereby mediating the polarization of M2 macrophages to exert anti-inflammatory effects and improve the bioavailability of BRB.