| Research background and purpose:Mercury（Hg）and its compounds are a kind of environmental pollutants and toxic substances in the world.With persistence and high bioaccumulation,mercury（Hg）can cause a series of biochemical changes in organisms,oxidative stress,inflammation and autophagy,which can directly or indirectly lead to various diseases in animals and humans.Mercuric chloride（HgCl2）can cross the blood-brain barrier and cause damage to the brain.It is known as one of the most neurotoxic heavy metals.Although it is not fat-soluble,mercuric chloride（HgCl2）can still cross the blood-brain barrier and accumulate in the nerve tissue and cause damage.Puerarin（Pue）is an isoflavone compound isolated and extracted from puerarin.It has the functions of anti-inflammatory,autophagy regulation,anti-oxidation and anti-apoptosis.However,it has poor water solubility,poor absorption in vivo and low bioavailability after oral administration.Therefore,in order to solve the problems of low oral bioavailability of Pue,this study planned to use PLGA as the carrier material to prepare the Pue-PLGA nanoparticle（Pue-PLGA-nps）drug delivery system,evaluate its preparation,and construct the brain injury model of HgCl2poisoned mice by gavage.To explore the biological effects of Pue and Pue-PLGA-nps on HgCl2-induced brain injury,so as to provide a theoretical basis for the wide clinical application of Pue and Pue-PLGA-nps and the prevention and treatment measures of HgCl2poisoning.Materials and Methods:1.Pue-PLGA-nps were prepared according to the optimal material ratio obtained in the previous response surface experiments of our research group.The pharmacologic evaluation was carried out in terms of particle size,zeta potential,drug loading,encapsulation rate and morphology under scanning electron microscope.The in vitro release characteristics of Pue-PLGA-nps were investigated by dialysis method,and SPF KM mice were used as the animal model.The in vivo metabolic characteristics of Pue-PLGA-nps were evaluated by HPLC.2.A mouse brain injury model of HgCl2poisoning was established by gavage,and Pue and Pue-PLGA-nps were used for intervention.The mice were divided into 5groups after 7 days of feeding:Normal saline group（NS）,HgCl2group（4 mg/kg）,Pue-PLGA-nps group（50 mg/kg）,HgCl2+Pue group（4 mg/kg+30 mg/kg）,HgCl2+Pue-PLGA-nps group（4 mg/kg+50 mg/kg）,and the test period was 28 days.The changes in body weight of the mice were recorded and the weight gain rate was calculated.The behavioral changes of the mice were observed by Morris water maze and balance beam experiments.The blood of the mice was collected after execution,the brains were weighed and recorded,and the brain-to-body ratio was calculated.The pathological changes of the brain tissues were observed by paraffin sectioning and H&E staining.The oxidative stress indicators（T-AOC,SOD,MDA,GSH-Px）in the brain tissues were measured.The levels of inflammatory factors（IL-6,IL-1β,TNF-α）in mice serum were determined by ELISA.TLR4,TRIM32,LC3 expression levels in brain tissues were measured by Westem blot and immunohistochemistry.3.Mice urine was collected using a metabolic cage 12 h before execution,mice blood was collected after execution,and brain was collected and weighed and recorded.The inductively coupled plasma mass spectrometry（ICP-MS）method for detecting Hg content in blood,urine and brain tissues of mice was first evaluated,and then the Hg content in blood,urine and brain tissues of mice was measured separately.Result:1.Pue-PLGA-nps was successfully prepared with a particle size of（121.1±0.9074）nm（PDI=0.084±0.011）,which was suitable and uniformly distributed,with a zeta potential of（-25.9±1.27）m V,an encapsulation rate of（85.87±0.51）%and a drug loading of（48.67±2.08）%,and SEM observation The morphology of Pue-PLGA-nps dry powder Pue powder particles were obvious,and the morphology of suspension showed rounded appearance and low breakage,which could be used effectively in the experiment.The in vitro release Results showed that the release rate of Pue was significantly higher than that of Pue-PLGA-nps under the same conditions,with a cumulative release rate of（97.96±0.93）%at 6 h,while the 6 h release rate of Pue-PLGA-nps was only（36.54±5.11）%,and the drug concentration of Pue-PLGA-nps rose to a certain value at the beginning of the release phase of Pue-PLGA-nps After that,it entered the stable release phase on the fifth day with a release rate of（82.17±2.05）%,decreasing release rate and long release time.The in vitro release curve fitted the Weibull equation to the highest degree,with a fitted equation of lnln[1-（1-Q）]=0.44394lnt-1.3204（R2=0.9777）.The Results of in vivo metabolism experiments showed that Pue concentrations in plasma and in brain tissue were higher in the Pue-PLGA-nps group than in the Pue group at different sampling time points.The calculation of pharmacokinetic parameters showed that in plasma,the AUC（0-t）,AUC（0-∞）,Cmax,tmax,t1/2,MRT（0-t）,MRT（0-∞）of Pue-PLGA-nps were 1.929,2.217,1.568,0.875,1.703,1.196,1.687 times higher than those of Pue 1.687 times;while in brain tissue,the AUC（0-t）,AUC（0-∞）,Cmax,tmax,t1/2,MRT（0-t）,MRT（0-∞）of Pue-PLGA-nps were 1.973,2.088,1.282,1.400,2.295,1.511,and 1.844 times higher than those of Pue,respectively.2.A mouse brain injury model induced by HgCl2was successfully established,and the intervention with Pue and Pue-PLGA-nps was able to improve the decrease of body weight gain and increase of brain-to-body ratio in mice caused by HgCl2,reverse the alteration of learning and memory ability and balance ability in mice caused by HgCl2,and reduce the pathological damage of brain tissue in mice caused by HgCl2.Meanwhile,Pue and Pue-PLGA-nps could not only attenuate HgCl2-induced oxidative stress damage in mouse brain tissue by down-regulating MDA levels and up-regulating the expression of T-AOC,SOD,and GSH-Px,but also attenuate HgCl2-induced inflammatory response by down-regulating the expression of pro-inflammatory cytokines IL-6,IL-1β,and TNF-α.Further studies revealed that Pue and Pue-PLGA-nps down-regulated the expression of inflammatory regulators（TLR4）,autophagy-related protein（LC3）and TRIM32.The experimental results showed that Pue and Pue-PLGA-nps had a protective effect on HgCl2-induced brain injury in mice,and the intervention effect of Pue-PLGA-nps was superior to that of Pue.3.The method established in this study for the determination of Hg in mouse blood,urine and brain tissue by ICP-MS showed a correlation coefficient of R=0.9989in the linear range of 0μg/L～5μg/L.The detection limit of the instrument was 0.017μg/L and the quantification limit was 0.056μg/L,which satisfied the requirements of the determination.The mean value of 3μg/L quality control solution was 2.99μg/L with an RSD of 0.5%,indicating the high stability of the instrument during the determination process.In the spiked recovery test,the Results for the three concentrations of low,medium and high（0.5,1 and 3μg/L）were in the range of95%～105%,indicating that the method has good accuracy and precision.The Results of sample determination showed that long-term exposure to HgCl2could cause the accumulation of Hg in brain tissues,blood and urine of mice;the intervention with Pue and Pue-PLGA-nps could reduce the Hg content in brain tissues,blood and urine of mice,and the experimental Results showed that the intervention effect of Pue-PLGA-nps was better than that of Pue.Conclusion:1.The Pue-PLGA-nps prepared in this study has good performance and has a certain slow-release effect,which can enhance the absorption of drugs in the body and lay the experimental foundation for the development of new oral formulations of Pue.2.HgCl2exposure induces brain injury in mice,and Pue-PLGA-nps can alleviate HgCl2-induced oxidative stress,inflammatory response,autophagy,and promote Hg excretion in mice through TLR4/TRIM32/LC3 pathway,and the intervention effect is better than Pue,and the successful preparation of Pue-PLGA-nps provides a new drug formulation for the clinical treatment of HgCl2exposure-induced brain injury. |
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