| Background and objectivesCerebrovascular disease (CVD) is one of the leading causes of death and disability throughout the world. Of the subtypes, ischemic cerebrovascular disease (ICD) accounts for80%of the disability and death. However, lack of specific and effective drugs is still a major problem for the treatment of ICD at present.Puerarin has been proved to have protective effects on cerebrovascular ischemia-reperfusion injury (CIRI), and applied in China for the treatment of cerebrovascular and cardiovascular diseases. However, the poor oral bioavailability and low drug concentration in brain limit its clinical apppliaction. Acetylpuerarin (AP), a kind of derivant of pueratin, has been proved in our previous studies to have protective effects against CIRI by reducing damage of oxygen-free radicals, increasing the activity of antioxidase, decreasing calcium concentration in neuron and inhibiting cell apoptosis in ischemia area in vivo.However, whether AP can permeate blood brain barrier (BBB) and how it acts in vivo are not known yet.In recent years, nanoparticles (NPs) have been used widely to deliver drugs to the brain, due to their ability of controlled drug release and site-specific drug-targeting. NPs have a further advantage over larger bulk materials, because they have a higher surface-to-volume ratio and therefore the dose and frequency of administration are reduced. Moreover, NPs can protect the loaded drugs from being biodegraded or pumped out of cells by P-glycoprotein (P-gp). Poly (lactic-co-glycolic acid)(PLGA) is one of the most successfully used biodegradable polymers because of its metabolite monomers, lactic acid and glycolic acid. The two monomers are endogenous and easily metabolized via the Krebs cycle, so a minimal systemic toxicity is attained with the use of PLGA for drug delivery. Therfore, PLGA is approved by the US FDA in lots of drug delivery systems in human.So in this study, we aim to develop polysorbate80coated acetylpuerarin-loaded PLGA nanoparticles to achieve high drug concentration in brain, study the pharmacokinetics and tissue distribution of the prepared nano-dilivery system in animals, and evaluate its protective effects on the injury of focal cerebral ischemia-reperfusion in rats.Inflammatory response is one of the major mechanisms that lead to CIRI. Inflammatory cytokines such as tumor necrosis factor a (TNF-a) and interleukin-1β (IL-1β) as well as adhesion molecules (including ICAM-1and VCAM-1) produced at the early stage of ischemia are involed in the secondary damage of cerebral ischemia-reperfusion. So anti-inflammatory therapies are beneficial to ischemic cerebrovascular disease outcome.High-mobility group box-1(HMGB1) protein is originally described as a nonhistone DNA-binding protein in the nucleus of eukaryotic cells, and has been implicated in the stabilization of nucleosomal structure and the facilitation of gene transcription. Recently, HMGB1emerges as a key cytokine to play an extracellular role when it is involved in cellular activation and proinflammatory responses. HMGB1can be actively released from immune cells (such as macrophages, NK cells, dendritic cells, and endothelial cells) and passively released from the necrotic or damaged cells. Once released into the extracellular milieu, HMGB1can bind to cell surface receptors (including advanced glycation end products (RAGE) and toll-like receptors like TLR2and TLR4) and activate the nuclear factor κB (NF-κB), which induces the up-regulation of leukocyte adhesion molecules and the production of proinflammatory cytokines, thereby, promoting inflammation.HMGB1is widely expressed in the nucleus of neurons, astrocytes, and pituicytes in the brain of mice. Many studies have proved that HMGB1plays an important role in the diseases of central nervous system (CNS), it can mediate neuritic outgrowth, regulate neuroendocrine response and glutamate release. In addition, HMGB1has been implicated in the mechanisms of ischemic brain damage. Studies have revealed that HMGB1is massively released into the extracellular space immediately after ischemic insult from neurons and subsequently induces the release of inflammatory mediators in the post-ischemic brain. Furthermore, oxygen glucose deprivation, which mimics ischemia in vitro, induces the release of HMGB1in the culture media of neurons. HMGB1, in turn, increases the levels of proinflammatory cytokines such as TNF-a and IL-lp in brain, which further stimulate the release of HMGB1and promote the injure of the post-ischemic brain. So HMGB1acts as a key regulator in the inflammatory process of ischemic brain damage.Our previous studies have revealed the anti-inflammatory effects of AP in the post-ischemic brain. AP can inhibit the expression of the inflammatory cytokines such as TNF-a, IL-6and IL-1β and adhesion molecules including ICAM-1and VCAM-1. It can also inhibit the expression of NF-κB (p65) induced by lipopolysaccharide, and prevent its blinding the DNA. However, as an upstream element of the signal pathway of "HMGB1-TLR4-NF-KB", whether and how HMGB1is regulated by the drug of AP are not known yet. So another objective of the work is to observe the effects of AP on the expression of HMGB1and study its anti-inflammatory mechanisms based on the HMGB1-TLR4-NF-κB pathway.Methods1. Development and evaluation of acetylpuerarin-loaded PLGA nanoparticles(1)The method of solvent diffusion was used to prepare the polysorbate80coated acetylpuerarin-loaded PLGA nanoparticles (AP-PLGA-NPs).(2)The method was optimized with an homogeneous design combined with response surface methodology following single factor study. The encapsulation efficiency (EE) and drug loading (DL) of AP in the NPs were evaluated based on the main factors including PLGA concentration, volume ratio of aqueous phase to oil phase and AP feeding amount.(3)The prepared AP-PLGA-NPs with the optimized method were evaluated by the characterization of morphological characteristics, particle size distribution, zeta-potential, in vitro release profile and preliminary stability.2. Pharmcokinetics of acetylpuerarin-loaded PLGA nanoparticles in rats(1)The method of liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was developed and validated by assessing the sample preparation, the conditions of chromatography and mass spectrometry, specificity, calibration curve, precision, recovery and matrix effects.(2)The plasma concentrations of AP and its metabolite puerarin (PUE) in rats were determined with the developed LC-MS/MS method, following intravenous administration of AP solution and AP-PLGA-NPs.(3)The pharmacokinetic parameters of AP and PUE were calculated with the software of Drug and Statistics (DAS,version2.1.1), and the differences of the parameters between the groups of AP solution and AP-PLGA-NPs were evaluated wih the software of SPSS (version19.0).3. Tissue distribution of acetylpuerarin-loaded PLGA nanoparticles in mice(1)The method of high performance liquid chromatography (HPLC) was developed and validated by assessing the sample preparation, the conditions of chromatography, specificity, calibration curve, precision and recovery.(2)The concentrations of AP and PUE in different tissues were determined with the developed HPLC method, and the tissue distributions of AP and PUE were observed, following intravenous administration of AP solution and AP-PLGA-NPs.(3)The brain-targeting characters of AP-PLGA-NPs were assessed based on the targeting parameters including overall targeting efficiency(Te), relative targeting efficiency (Re) and ratio of peak concentration (Ce).4. Effects of acetylpuerarin-loaded PLGA nanoparticles on the injury of focal cerebral ischemia-reperfusion in rats(1)The model of brain ischemia-reperfusion injury was prepared by a method of transient middle cerebral artery occlusion (MCAO) in rats according to the report of Zea Longa. The MCAO rats were produced by intraluminal occlusion of the left middle cerebral artery with a nylon monofilament.(2)Healthy clean male Wistar rats were randomly divided into six groups:sham group, ischemia/reperfusion (I/R) group, I/R+AP solution at the dose of15mg/kg (AP-L) group, I/R+AP solution at the dose of30mg/kg (AP-H) group, I/R+AP-PLGA-NPs at the dose of15mg/kg (AP-NPs) group, and I/R+ethyl pyruvate solution at the dose of40mg/kg (EP) group. All drugs were administered via tail-vein injection0.5h before ischemia and at the onset of reperfusion.(3)The effects of AP on cerebral infarction volume, neurological outcome, pathological issue change and apoptosis of nerve cells were observed following I/R injury in rats. The neurological deficit scores were assessed according to Zea Longa’s method, the cerebral infarction size was measured by triphenyltetrazolium chloride (TTC) staining, pathological changes of ischemic hippocampus and cortex were observed by hematoxylin-eosin (HE) staining, and the number of apoptotic neurons in the ischemic cortex was counted following TUNEL staining.5. Effects of acetylpuerarin on the protein expression of HMGB1and its anti-inflammatory mechanism based on the HMGB1-TLR4-NF-κB pathway(1) The oxygen-glucose deprivation and reperfusion injury in BV-2cells was developed to mimic cerebral ischemia reperfusion injury in animals. BV-2cells were divided into the following groups:normal group, OGD group, treatments with different doses of AP(0.1、0.4、1.6μM) and EP(1mM) groups.The viability of cells was determined by CCK-8(Cell Counting Kit-8) assay, the apoptosis in BV-2cells was assessed by Hoechst33258and Annexin-V/7-AAD staining, and the effects of AP on the nuclear release of HMGB1induced by OGD were observed by green immunofluorescent FITC-staining.(2) Dynamic expression of HMGB1in rats after reperfusion:The protein expressions of HMGB1in the ischemic cortex of rats were analyzed by western blot method, and the serum concentrations of HMGB1were determined by enzyme-linked immunosorbent assay (ELIS A) at different time after reperfusion. (3) Effects of AP on the protein expression of HMGB1, TLR4and NF-κB (P65)Healthy clean male Wistar rats were randomly divided into five groups:sham group, ischemia/reperfusion (I/R) group, I/R+AP solution at the dose of15mg/kg (AP-L) group, I/R+AP solution at the dose of30mg/kg (AP-H) group, and I/R+ethyl pyruvate solution at the dose of40mg/kg (EP) group. All drugs were administered via tail-vein injection0.5h before ischemia and at the onset of reperfusion.The protein expression of cytoplasmic HMGB1, total TLR4and nuclear NF-κB (P65) were assessed by western blot method, the protein expression and transposition of HMGB1and NF-κB (P65) were observed by immunohistochemistry assay, and serum concentration of HMGB1were determined by ELISA. The effects of AP on the protein expression of cytoplasmic HMGB1, total TLR4and nuclear NF-κB (P65) were evaluated24h after reperfusion. Results1. Development and evaluation of acetylpuerarin-loaded PLGA nanoparticles(1)Considering all the factors including higher DL, higher EE and AP solubility, the optimum results based on the homogeneous design combined with response surface methodology were as follows:PLGA concentration=18mg/mL, volume ratio of aqueous phase to oil phase=4, AP feeding amount=7mg. The experimental results indicate that response surface methodology can well predict the preparation for AP-PLGA-NPs.(2)The prepared AP-PLGA nano-system was a blue opalescent colloidal solution, exhibiting a spherical shape under transmission electron microscopy with an average size of145.0nm and zeta potential of-14.81mV. The polydispersity index (PI) of the prepared NPs was0.153, the EE and DL was90.51%and17.07%, respectively.(3)The in vitro release of AP-PLGA-NPs showed an initial burst release followed by a sustained release. The control preparation of AP suspension was found to have higher r value for first-order kinetic model, while the release pattern followed Higuchi model for AP-PLGA-NPs. (4)The preliminary stability study showed that the storage of AP-PLGA-NPs at4℃could keep a better stability compared with the storage at room temperature.2. Pharmcokinetics of acetylpuerarin-loaded PLGA nanoparticles in rats(1)AP and the metabolite PUE were simultaneously detected in the plasma of rats following intravenous administration of AP formulations, while no AP but PUE was detected following oral administration.(2)The AUC0-∞values of AP and PUE following intravenous administration of AP-PLGA-NPs were5398.60±724.77and15106.98±3652.73ng-mL-1·h, which was2.90and2.29times as great as that of AP solution, respectively. Similarly, the t1/2values of AP and PUE were3.22±0.52and3.58±0.41h, which was2.19and1.98times as great as that of AP solution, respectively. While the Cmax values of AP and PUE showed no significant differences between AP-PLGA-NPs and AP solution groups.(3)The AUC0-∞and Cmax value of PUE after oral administration of AP-PLGA-NPs was6175.66±350.31ng·mL-1·h and1301.13±101.41ng·mL-1, which was2.75and1.78times as great as that of AP suspension, respectively, indicating enhanced drug absorption.3. Tissue distribution of acetylpuerarin-loaded PLGA nanoparticles in mice(1)The AUCo-∞of PUE following intravenous administration of AP solution was greatest in liver, followed by kidney, heart, lung, spleen and brain in turn. The tissue distribution of PUE was greatly changed due to the encapsulation of PLGA, and significantly increased AUC in brain and liver and prolonged MRT in brain were observed in AP-PLGA-NPs group, compared with AP solution.(2)AP and the metabolite PUE were simultaneously detected in the brain of mice following intravenous administration of AP formulations.(3)The overall targeting efficiency(Te) of PUE in brain was2.29%for AP-PLGA-NPs, which was much higher than that of1.28%for AP solution. The relative targeting efficiency (Re) in brain was2.40and2.58for AP and PUE, respectively. And the ratio of peak concentration (Ce) in brain was1.91and1.89for AP and PUE, respectively. The targeting parameters indicated that the distributions of AP and PUE in brain tissue were greatly changed due to the formulation of NPs.4. Effects of acetylpuerarin-loaded PLGA nanoparticles on the injury of focal cerebral ischemia-reperfusion in rats(1)The neurologic deficit scores were significantly higher in I/R group than those in sham group. Treatments with different dose of AP formulations significantly decreased the NDS (p<0.01), compared with I/R group. Moreover, lower NDS were found in AP-H (1.67±0.65, P<0.05) and AP-NPs group (1.42±0.51, P<0.01), compared with AP-L group (2.17±0.39).(2)The percentage of infarct volume in I/R group was40.14±2.61%. Treatments with different dose of AP formulations significantly decreased infarct volume (P<0.01), compared with I/R group. Furthermore, significant difference (P<0.01) in infarct volume was observed between AP-L (29.39±3.67%) and AP-NPs group (20.47±2.26%).(3)The results of HE staining showed that the ischemic hippocampus and cortex tissues were damaged seriously in I/R group. Neurons were degenerated and necrotic, and their arrangement was disordered. Treatments with AP formulations greatly decreased the pathological damage and markedly increased the amount of normal neurons. Moreover, the effects of AP-NPs on the ischemic damage appeared more obvious, compared with AP-L and AP-H groups.(4)The results of TUNEL staining showed that the number of TUNEL positive cells in the ischemic cortex in I/R group significantly increased, compared with sham group. Treatments with AP formulations greatly decreased the number of TUNEL positive cells. Furthermore, TUNEL positive cells were significantly decreased in AP-NPs group, compared with AP-L group.5. Effects of acetylpuerarin on the protein expression of HMGB1and its anti-inflammatory mechanism based on the HMGB1-TLR4-NF-κB pathway(1)The results of CCK-8assay indicated that different doses of AP significantly improved the viability of cells, and the results of Hoechst33258and Annexin-V/7-AAD staining showed that AP greatly inhibited the apoptosis in BV-2cells. Furthermore, AP attenuated the release of HMGB1and retained it in the nucleus in BV-2cells.(2) Dynamic expression of HMGB1in rats after reperfusion①The results of western blot showed that little HMGB1was expressed in cytoplasm, however the expression of cytoplastic HMGB1increased at12h, peaked at24h, and had been maiteined a higher level till72h after reperfusion (P<0.01), compared with sham group.②The results of ELISA indicated that serum concentration of HMGB1increased at12h after reperfusion, but no statistic differences were found (P>0.05), compared with sham group. The concentration of HMGB1peaked at24h, and had been maintained a higher level till72h after reperfusion (P<0.05), compared with sham group. The results of HMGB1concentration were coincident with those of western blot method(3) Effects of AP on the protein expression of HMGB1, TLR4and NF-κB (P65)①Treatments with different dose of AP greatly decreased the expression of cytoplasmic HMGB1, total TLR4and nuclear NF-κB (P65)24h after reperfusion (P<0.01), compared with sham group. The effects of AP showed dose-depended.②The serum concentrations of HMGB1in AP-L and AP-H group were greatly decreased (P<0.01), compared with sham group, but there was no significant difference among AP-L, AP-H and EP groups.③Few cells were stained with HMGB1and NF-κB (P65) in the ischemic cortex in sham group by immunohistochemistry, which were found in nuclei and cytoplasm, respectively. The number of HMGB1and NF-κB (P65) positive cells increased in the ischemic cortex in I/R group (P<0.01), which were found in both cytoplasm and nuclei. Treatments with AP decreased the number of HMGB1and NF-κB (P65) positive cells, and inhibited the translocation of HMGB1from nucleus to cytoplasm, and NF-κB (P65) from cytoplasm to nucleus, respectively. Conclusions1. The polysorbate80coated acetylpuerarin-loaded PLGA nanoparticles prepared with optimized solvent diffusion method were uniform in particle size and showed sustained-release in vitro.2. Acetylpuerarin was metabolized to puerarin in vivo, both acetylpuerarin and puerarin were detected following intravenous administration. The PLGA nanoparticles greatly changed the pharmacokinetic profiles of acetylpuerarin and puerarin, and significantly increased the drug distributions in the brain tissue, showing brain-targeting character.3. Acetylpuerarin showed protective effects against oxygen-glucose deprivation-reperfusion damage in BV-2cells and focal cerebral ischemia-reperfusion injure in rats following intravenous administration; the effects of drug loaded nanoparticles appeared more obvious, due to the sustained-release and brain-targeting characters; it was the combined actions of acetylpuerarin and puerarin that contributed to the brain-protective effects.4. No acetylpuerarin but puerarin was detected in rat plasma after oral administration. The PLGA nanoparticles greatly enhanced the oral bioavailability of acetylpuerarin.5. Acetylpuerarin decreased the protein expression of HMGB1, TLR4and NF-κB (P65), and inhibited the nuclear release of HMGB1and translocation of NF-κB (P65) from cytoplasm to nucleus, which contributed to its anti-inflammatory effect. |
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