Lisuride is a G protein biased agonist at the 5-HT2AR and, unlike the structurally-related lysergic acid diethylamide (LSD), the drug does not typically produce hallucinations in normal subjects at routine doses. Here, we examined behavioral responses to lisuride, in wild-type (WT), βArr1-knockout (KO), and βArr2-KO mice. In the open field, lisuride reduced locomotor and rearing activities, but produced a U-shaped function for stereotypies in both βArr lines of mice. Locomotion was decreased overall in βArr1-KOs and βArr2-KOs relative to wild-type controls. Incidences of head twitches and retrograde walking to lisuride were low in all genotypes. Grooming was decreased in βArr1 mice, but was increased then decreased in βArr2 animals with lisuride. Serotonin syndrome-associated responses were present at all lisuride doses in WTs, but they were reduced especially in βArr2-KO mice. Prepulse inhibition (PPI) was unaffected in βArr2 mice, whereas 0.5 mg/kg lisuride disrupted PPI in βArr1 animals. The 5-HT2AR antagonist MDL100907 failed to restore PPI in βArr1 mice, whereas the dopamine D2/D3 antagonist raclopride normalized PPI in WTs but not in βArr1-KOs. Clozapine, SCH23390, and GR127935 restored PPI in both βArr1 genotypes. Using vesicular monoamine transporter 2 mice, lisuride reduced immobility times in tail suspension and promoted a preference for sucrose that lasted up to 2 days. Together, it appears βArr1 and βArr2 play minor roles in lisuride’s actions on many behaviors, while this drug exerts anti-depressant drug-like responses without hallucinogenic-like activities.There is now evidence from multiple Phase II clinical trials that psychedelic drugs can exert long-lasting anxiolytic, anti-depressant, and anti-drug abuse (nicotine and ethanol) effects in patients. Despite these benefits, the hallucinogenic actions of these drugs at the serotonin 2A receptor (5-HT2AR) limit their clinical use in diverse settings. Activation of the 5-HT2AR can stimulate both G protein and  Parenthignaling . Recentlin vitro  and in v. With lisuride, locomotion in both genotypes decreased in a dose-dependent fashion from 0.01 to 0.5 mg/kg lisuride relative to vehicle  and remained flat.When cumulative locomotor activities were examined at baseline in βArr1 animals, no significant genotype or treatment effects were found . Followip = 0.006) and their assigned treatment groups to be different (p < 0.001)  and remained low to the 4 mg/kg dose.An examination of cumulative rearing found basal responses between βArr1 genotypes (< 0.001) . To contp = 0.003)  and then these activities increased with 4 mg/kg lisuride but were still below that of vehicle (p < 0.008).Basal cumulative stereotypies were different among βArr1 mice prior to treatment (= 0.003) . While np < 0.001). All doses of lisuride suppressed locomotion compared to vehicle .Motor activities were evaluated also in βArr2 mice. No genotype or treatment effects were discerned for baseline cumulative locomotion . Nonethep < 0.001) .An assessment of cumulative rearing in βArr2 animals revealed basal responses to be different across treatment assignments (< 0.001) . The ANCp = 0.042) and treatment assignment (p < 0.001) (p = 0.056) where responses were lower overall in βArr2-KO than WT mice. Treatment effects were more dramatic and were U-shaped. Here, the numbers of cumulative stereotypies declined from vehicle to 0.01 lisuride (p < 0.001), responses were low to 2 mg/kg , then ascended with 4 mg/kg lisuride to the vehicle control.Basal cumulative stereotypies were differentiated by genotype (< 0.001) . FollowiTaken together, these findings show that in both βArr1 and βArr2 mice lisuride reduces cumulative locomotor, rearing, and stereotypical activities; however, these latter activities assume a biphasic U-shaped function with lisuride.p < 0.001) . Importantly, the numbers of responses with 4 mg/kg lisuride were enhanced over all other lisuride doses . In βArr1-KO mice the incidences of 5-HT syndrome-associated responses were highest also with 4 mg/kg relative to vehicle and 0.01, 0.05, 0.5, and 1 mg/kg lisuride ; responses to 2 mg/kg lisuride were increased over vehicle and the 0.5 mg/kg dose .To determine which behaviors were expressed as stereotypies, responses were scored over 30 min immediately following lisuride administration. We identified many behaviors associated with 5-HT syndrome and they consisted of hunched posture, repetitive paw kneading, rigid posture, flat posture, Staub tail, and tremor or body shaking see . HTRs an< 0.001) . Within p-values≤0.001)  and 0.1–4 mg/kg lisuride produced more responses than the 0.01 mg/kg dose . In addition, responses to 0.1 and 4 mg/kg lisuride were higher than for the 0.05 mg/kg dose . In βArr2-KO animals, responses to the 1 and 4 mg/kg doses were enhanced over vehicle and 0.01 mg/kg lisuride . The 4 mg/kg lisuride-induced responses were higher also than to 0.01 and 0.1 mg/kg lisuride . Collectively, these results show that 5-HT syndrome-associated responses are elicited at much higher rates in WT than βArr2-KO mice to 0.05–4 mg/kg lisuride, while only the highest lisuride dose augments more responses in WTs than βArr1-KO animals.Genotype differences were robust in βArr2 mice, where all doses but 0.01 mg/kg lisuride stimulated the expression of more 5-HT syndrome-associated responses in WT than βArr2-KO animals (s≤0.001) . Within p-values≤0.004). Even with this high 4 mg/kg dose less than 7 HTRs were identified over the 30-min post-injection period, whereas LSD stimulated high levels of HTRs relative to all other treatments . In βArr2 animals, the numbers of HTRs barely exceeded 3 with 4 mg/kg lisuride and this number was not significantly different from vehicle (p < 0.001). Thus, with lisuride HTRs are exceedingly low in both βArr lines, while LSD stimulates more HTRs in βArr1-KO and WTs than βArr2-KO mice.When HTRs were examined, both WT and βArr1-KO mice responded similarly to lisuride . Here 4  vehicle . By contp-values ≤0.030) (p = 0.021) and it remained high to 0.5 mg/kg lisuride, then declined with the highest dose (p = 0.006).For self-grooming, no genotype effects were detected in either βArr strain . Compare ≤0.030) . By cont ≤0.030) . From a p-values<0.001), whereas 2 and 4 mg/kg lisuride augmented the response from the 0.5 and 1 mg/kg doses . With βArr2 mice, a biphasic dose-response curve was found (p = 0.025), while retrograde events were increased from this nadir with 4 mg/kg lisuride.For retrograde walking, no genotype effects were found with lisuride . Retrogras found . DeclineCollectively, only treatment effects with lisuride are obtained for HTRs, grooming, and retrograde walking. Overall HTRs are higher in βArr1 than βArr2 mice, but they are very low compared to LSD. The duration of grooming is decreased in βArr1 mice, but is enhanced in βArr2s except at 4 mg/kg lisuride. By contrast, the incidences of retrograde walking are represented by a U-shaped function in both mouse strains.p-values ≤0.007). Hence, subsequent experiments were conducted with 0.5 mg/kg lisuride and different receptor antagonists to determine if they could normalize PPI.LSD- and certain other psychedelic-induced states share many similarities with early acute phases of psychosis . PPI is p-values<0.001), the antagonist failed to normalize PPI (p < 0.001).The 5-HTR2A antagonist, MDL100907, was tested first in βArr1 animals . No genop = 0.018) .The dopamine D2/D3 (D2/D3) antagonist, raclopride, was evaluated next. Here, the lisuride disruption of PPI in WT mice was normalized with 3 mg/kg raclopride (= 0.018) . In βArrp-values≤0.001). As lisuride binds to dopaminergic, adrenergic, and various serotonergic receptors (p < 0.001) (p = 0.001)   and 2 mg= 0.001) . In cont= 0.001) .Besides PPI, null and startle activities were examined in both βArr1 and βArr2 mice where some genotype and treatment effects were noted . NeverthIn summary, PPI was disrupted in βArr1 mice given 0.5 mg/kg lisuride and MDL100907 was unable to reverse this effect. Raclopride normalized PPI but only in WT mice; PPI in βArr1-KOs remained abnormal. Clozapine, SCH23390, and GR127935 restored PPI in both WT and βArr1-KO mice. In βArr2 mice, PPI was retained with all lisuride doses.WT and VMAT2-HET mice were used to assess anti-depressant drug-like actions of lisuride. Parenthetically Fukui and colleagues (2007) reported adult VMAT2-HETs were hypoactive, displayed anhedonia-like responses, and showed enhanced immobility in tail suspension and forced swim that were normalized with tricyclic anti-depressants, selective 5-HT and norepinephrine transporter inhibitors, and bupropion. These mutants were more responsive to stress and displayed enhanced learned helplessness compared to WTs.p < 0.001), whose preference was similar to day 1 W-W. Following acute administration of 0.5 mg/kg lisuride on day 3, the VMAT2-HETs’ sucrose preference exceeded WTs (p = 0.003) and it was significantly higher from their selections on days 1 and 2 . On day 4, the mutants’ preference was similar to WT mice. However, by days 5 and 6 sucrose preference in VMAT2-HET mice fell from WT levels  where their preference was similar to day 1 W-W. One reason why sucrose preference could vary across days in mutants might be due to differences in intake. However, no genotype differences were found across days . Collectively, VMAT2-HET mice fail to show a sucrose preference until they are given lisuride. This preference persists for 2 days and declines thereafter, whereas sucrose preference is maintained in WTs regardless of lisuride treatment. Importantly, these differences in sucrose preference cannot be attributed to differential fluid consumption between genotypes.WT and VMAT2-HET mice were tested on day 1 with water-water (W-W) pairings where they showed no bottle or side-preference . On day oss days . Nonethep-values≤0.006). Additionally, immobility with LSD was higher than in fluoxetine and 0.5 mg/kg lisuride groups . On days 3 and 5 no treatment effects were present. In VMAT2-HETs acute administration of fluoxetine, LSD, or 0.5 mg/kg lisuride reduced immobility times compared to vehicle   and those for LSD were decreased relative to 1 mg/kg lisuride (p = 0.014). By days 3 and 5, all treatment effects had vanished. Hence, 0.5 mg/kg LSD and lisuride were efficacious acutely, while fluoxetine reduced immobility in VMAT2-HETs up to 24 h after administration.In tail suspension (6 min), VMAT2 mice were treated acutely with vehicle, 20 mg/kg fluoxetine, or 0.5 or 1 mg/kg lisuride and tested 30 min later. With 0.5 mg/kg LSD, mice were tested acutely 2 h after administration to ensure the motor-activity stimulating effects of LSD were no longer evident see . Subsequs≤0.043) . Effectsp-values ≤0.020) and immobility in 0.5 mg/kg lisuride VMAT2-HETs was lower than its vehicle (p = 0.042). Twenty-four hr post-injection (day 1) immobility was reduced in vehicle-vehicle versus reserpine-vehicle mice and in both VMAT2-HET groups . Additionally on day 1, immobility in reserpinized animals was higher than in reserpine plus lisuride mice and both VMAT2-HET groups . On days 3 and 5, immobility times were decreased in vehicle-vehicle mice compared to all other groups . In summary, immobility times in tail suspension were lowest in C57BL/6 J vehicle-vehicle controls across days, whereas 0.5 mg/kg lisuride showed anti-depressant like activities in VMAT2-HET and reserpinized C57BL/6 J mice, respectively, acutely and 24 h post-injection.Anti-depressant like activities were evaluated also in the reserpinized mouse model as VMAT2 is a target for this drug . C57BL/6versus vehicle (p < 0.001)  . A simil< 0.001) . Thus, tIn summary, the tail suspension and sucrose preference results reveal that 0.5 mg/kg lisuride possesses anti-depressant drug-like actions that last acutely or at least over 24 h.Locomotor, rearing, and stereotypical activities have been studied in various strains of rats—primarily males—and different strains of male and female mice. Lisuride most commonly produced a biphasic locomotor response where low doses decreased and higher doses were stimulatory . Other rIn rats and mice, lisuride decreased rearing in the open field and BPM  and our To identify behaviors represented in stereotypy, responses were scored over the first 30 min post-injection interval. Behaviors associated with the 5-HT syndrome appeared in the βArr lines with the lowest lisuride dose and they consisted primarily of hunched posture, paw kneading, rigid posture, flat posture, Staub tail, and tremor or body shaking see . Some ofHtr2a in mice abrogates this response  cells used for DNA injection, different strains of mice used to implant the ES cells, and the mouse strains used for outcrossing the mutations . With respect to lisuride, there is evidence it may have anti-depressant actions in post-stroke patients experiencing depression  and it was brought to volume with 5% 2-hydroxypropoyl-β-cyclodextrin (Sigma-Aldrich) in sterile water . Clozapine was solubilized with glacial acetic acid  and was brought to volume with 5% 2-hydroxypropyl-β-cyclodextrin in sterile water. Fluoxetine was solubilized in sterile water. All drugs were injected (i.p.) in a 5 ml/kg volume.The drugs consisted of lisuride maleate (#4052), clozapine (#0444), SCH23390 (#0925), GR127935 , LSD-tartrate , fluoxetine HCl (#F132) and reserpine . The vehicle for lisuride, LSD, MDL, reserpine, SCH23390, and GR127935 consisted of The open field consisted of a clear Plexiglas arena 21 × 21 × 30 cm  illuminated at 180 lux . OverheaThese behaviors were filmed during the test for motor activity . All resPPI of the acoustic startle response was monitored in SR-LAB chambers  . Mice wead libitum, while the water bottle was removed 2.5 h from the home-cage before the beginning of the dark cycle. A pair of metering bottles  was filled with RO/Milli-Q water and the procedure was repeated until water consumption was stable and no side-bias evident. Subsequently, each pair of bottles was filled with water (W-W) or 0.5% sucrose (S-W). Note, the solution was prepared just prior to testing, the bottles were weighed before and immediately after testing, and they were cleaned and drained thoroughly each day after testing. The pair of bottles was presented to mice 1.5 h into their dark cycle  and they were allowed to drink for 2 h. Subsequently, bottles were removed and the original water bottle was returned to the home-cage at least 1 h later. The final W-W pairing occurred on day 1. The next day, animals were presented with the S-W pairing. On day 3 they were administered 1 mg/kg lisuride (i.p.) and 5 min later were given S-W. Subsequent S-W pairings were presented over days 4–6. The total volume of liquid consumed in 2 h was determined each day. Sucrose preference was calculated by dividing the volume of sucrose consumed by the total volume of liquid consumption.VMAT2 mice were housed individually for 7 days prior to and throughout the study . Food waVMAT2 mice were administered vehicle, 20 mg/kg fluoxetine, or 0.5 or 1 mg/kg lisuride and tested 30 min later see . When mip < 0.05 was considered statistically significant. The results displayed in all figures were plotted using GraphPad Prism 10.0.2 .Results are presented as means ±SEMs; all statistics were performed with SPSS 27 and 28 programs . Univariate ANOVAs analyzed treatment effects. If cumulative baseline motor activities were significantly different for genotypes or treatment assignments, the cumulative post-administration results were analyzed by analysis of covariance. Repeated measures ANOVA were used for analysis of open field, PPI, sucrose preference, and tail suspension studies and, if significant, were followed by Bonferroni-corrected pair-wise comparisons. A


Objectives: Since the global broadcast of multidrug-resistant gram-negative bacteria is accelerating, the use of Polymyxin B is sharply increasing, especially in critically ill patients. Unsatisfactory therapeutic effects were obtained because of the abnormal physiological function in critically ill patients. Therefore, the determination of optimal polymyxin B dosage becomes highly urgent. This study aimed to illustrate the polymyxin B pharmacokinetic characteristics by defining the influencing factors and optimizing the dosing regimens to achieve clinical effectiveness.Methods: Steady-state concentrations of polymyxin B from twenty-two critically ill patients were detected by a verified liquid chromatography-tandem mass spectrometry approach. The information on age, weight, serum creatinine, albumin levels, and Acute Physiology and Chronic Health Evaluation-II (APACHE-II) score was also collected. The population PK parameters were calculated by the non-parametric adaptive grid method in Pmetrics software, and the pharmacokinetic/pharmacodynamics target attainment rate was determined by the Monte Carlo simulation method.Results: The central clearance and apparent volume of distribution for polymyxin B were lower in critically ill patients . Moreover, albumin (ALB) levels can be used to explain the variability in clearance, and age can be used to describe the variability in the apparent volume of distribution. For maintaining clinical effectiveness and lowering toxicity, 75 mg q12 h is the recommended dosing regimen for most patients suffering from severe infections.Conclusion: This study has clearly defined that in critically ill patients, age and ALB levels are potentially important factors for the PK parameters of polymyxin B. Since older critically ill patients tend to have lower ALB levels, so higher dosages of polymyxin B are necessary for efficacy. Over the last decade, more pathogenic bacteria have developed resistance, making many first-line antibiotics ineffective against infections . PolymyxIn this study, 22 adult critically ill patients were given intravenous polymyxin B administration, and a population pharmacokinetic (PPK) model was created by the non-parametric adaptive grid (NPAG) algorithm. The relationship between PK parameters and covariates  was analyzed by linear regression. A covariate model would be built for further confirmation. For finding optimal dosing regimens of polymyxin B, the Monte Carlo approach was performed to estimate the probabilities of target attainment (PTA) for clinical dosing regimens in the simulated group with modifying age and various ALB concentration ranges.We gather data from critically ill patients (≥18 years old) who were given intravenous drip polymyxin B treatment with blood concentration monitoring at the hospital from November 2021 to September 2022. The attending physician decided to administer polymyxin B. At the same time, patients who received continuous renal replacement therapy (CRRT) (n = 10) or extracorporeal membrane oxygenation (ECMO) (n = 2) at the time of blood collection were excluded. Patients’ information, including gender, age, WT, serum creatinine (Scr), CCR, ALB, APACHE-II, infection diagnosis, and pathogenic bacteria, were obtained from the electronic system for medical records. CCR was generated by adopting the Cockcroft—Gault equation with WT and age . The Eth-1 after collection, and the upper plasma after centrifugation was stored at −20°C.Patients were treated with polymyxin B, and plasma samples were collected ≥ 48 h after the treatment. Each patient contributed clinical samples, of which 37.5% (n = 24) were sampled with troughs (within 1 h before administration), 35.9% (n = 23) were sampled with peaks (within 1 h after the end of the infusion), and 26.6% (n = 17) were sampled at 6-8 h after the infusion. The dosing regimens of polymyxin B were a loading dose of 100 mg–150 mg and a maintenance dose of 50 mg–75 mg q12h, and the infusion duration was about 1–2 h. In preparation for analysis, samples were centrifuged for 10 min at 3000 r min-1, and the column temperature was set to 40°C. The parent/daughter ion mass ratios used in the mass spectrometry were m/z 602.6/241.2 (polymyxin B1 and B1-Ile) and m/z 595.6/202.1 (polymyxin B2). Plasma samples (100 μL) were collected from centrifuged blood samples, mixed with 500 μL precipitant (acetonitrile with 6.67% formic acid) and vortexed well for 1 min. The lower limit of quantification (LLOQ) of the bioanalytical method used for quantifying polymyxin B1, B2, and B1-Ile concentrations was 0.19, 0.02 and 0.02 mg L-1, respectively. The linear relationships of polymyxin B1, B2, and B1-Ile concentrations in the range of 0.19–24.13 mg L-1, 0.02–2.7 mg L-1 and 0.02–2.38 mg L-1 were considered good  and the accuracy was within 95.3%–105.3%, 92.2%–104.8%, and 95.8%–105.4%. The RSD (%) values for the intra- and inter-day precision (n = 5) were within 2.47%–5.21%, 1.98%–6.36%, and 3.15%–6.16%, and the recovery rates were 92.89%–106.19%, 86.16%–99.94%, and 90.01%–102.30%. Finally, 200 μL of the supernatant from the centrifugation of the combination at 14,000 r min-1 for 10 min was employed to measure the amounts of polymyxin B.According to previous studies, total polymyxin B1, polymyxin B2, and polymyxin B1-Ile concentrations in human blood plasma were quantified using LC-MS/MS . The sta0 + C1 × Cobs + C2× Cobs2 + C3 ×Cobs3, where C0, C1, C2, and C3 are scientifically set to 0.1, 0.15, 0, and 0, roughly. The gamma value at the start was set to 5. The model fit indexes  and the improvement in the goodness of fit plots  were the model’s evaluation metrics. A standard two-compartment PK model can be described in the model building process by the following differential Eqs In order to analyze the total concentrations of polymyxin B, Pmetrics version 1.9.7  was used by R version 4.1.3 . PK para2 test, p < 0.01, df = 1) or the population fit value (R2 pop) was improved from the initial model. The diagnostic plot of goodness-of-fit and the visual predictive check (VPC) were used for model visual assessment. The bootstrap method and the data partitioning (dividing the entire dataset randomly into 80% and 20% parts) were utilized to gauge the model’s stability and accuracy.Rate IV represents the rate of intravenous infusion. The central ventricular distribution volume (Vc) and central ventricular clearance (CL) were the fundamental PK parameters for the one-compartment model. In contrast, peripheral ventricular distribution volume (Vp) and inter-compartmental clearance (Q) were added for the two-compartment model. During covariate model construction, we evaluated covariates that might be associated with PK parameters, including age, WT, CCR, ALB, and APACHE-II score. The application of linear regression was to assess the strength of the correlations between variables and PK parameters. The covariate inclusion criteria were set as the amount of change in -2LL was more significant than 6.63 , the low ALB level group (25–34.9 g L-1), and the normal ALB level group (35 –41.5 g L-1). At the same time, the data has also been divided into three parts according to age, including the 5th percentile (34 years), median (68 years), and 95th percentile (93 years) values. We simulated the likelihood of achieving the AUC/MIC ≥ 50 or AUCss,24h > 100 mg∙h∙L-1 under various dosing regimens of polymyxin B by Pmetrics package (n = 1000). The regimens were set as follows: (Ⅰ) 100 mg q12 h; (Ⅱ) 75 mg q12 h; (Ⅲ) 60 mg q12 h; (Ⅳ) 50 mg q12h; (Ⅴ) 40 mg q12 h.Combined with population PK/PD feature parameters of antibiotics medicines, the PTA of PK/PD target for a dosing regimen at a predetermined minimum inhibitory concentration (MIC) was performed by Monte Carlo simulation. According to the idelines , the tar be 0.42 . Thus AU0.42  resistant to carbapenem antibiotics. Other statistical information about the patients was shown in .The study included twenty-two participants, with sixty-four blood concentration measurements. Most patients (95%) had lung or bloodstream infections, and nearly all of the microorganisms that caused those illnesses were bacteria (p < 0.01), and the changes in other indicators were shown in 2 = 0.17), as well as Vc and APACHE-II (R2 = 0.17), while a correlation was found between CL and ALB (R2 = 0.20), which was shown in 2 = 0.01) and WT with V (R2 = 0.01).One- and two-compartment models were considered, while -2LL was used to set the base model. For polymyxin B, compared to the one-compartment model, the two-compartment model performed better  and the R2pop value was enhanced to 0.573. As a result, model 6 was chosen as the final model. According to the last cycle, the gamma value was 0.72. The PK parameters of the final model for polymyxin B are shown in -1 and 16.64 ± 12.74 L. Finally, in the model 6, after the inclusion of age and ALB in the two-compartment model, the value of—2LL decreased > 6.63  was displayed in -1, dosing regimens should be selected according to different ALB and age. As long as the MIC is up to 1 mg L-1, patients in the ultra-low ALB levels group (23.1–24.9 g L-1) need to choose a dosing regimen of 100–75 mg q12 h  and V (16.64 L) (-1) (-1) (-1) (-1) (2 = 0.17) and defined age as a vital element for V of polymyxin B in the covariate model.Since small samples were employed in this experiment, the non-parametric population modeling algorithm has theoretical advantages over parametric methods in detecting population PK/PD subgroups and outliers , so the 16.64 L)  in this  L) (-1)  Table 3-1 and V  L) (-1) . However-1) (-1)  or under-1) (-1) . We assu-1) (-1)  while in-1) (-1) , possibl-1) (-1) . It is e-1) (-1) . HoweverEnterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii, MIC = 2 mg L-1 is determined as the mediator value, and MIC = 4 mg L-1 is determined as the resistance value. The MIC value of the pathogenic strain is also an essential factor that should be considered when utilizing polymyxin B for clinical therapy. The results of Monte Carlo simulation illustrate that, for MIC of polymyxin B against bacterial strain ≤ 1 mg L-1, 100–75 mg q12 h should be considered for patients with ultra-low ALB stages . Moreover, to figure out the risk of polymyxin B’s toxic effects, the PTA of different dosing regimens reaching renal damage AUCss, 24h (≥100 mg∙h∙L-1) were shown in -1 . In summary, we eventually advise a dosing regimen of 75 mg q12 h for most critically ill patients with MDR-GNB infections. Also, the dose of polymyxin B for critically ill older patients should not be decreased if their ALB levels are low or ultra-low.Since the MIC of polymyxins differs for each pathogen, for B stages , while 1B stages . What’s 93 years . HoweverB levels . The PTAwn in -1 , becauseHowever, there are still shortcomings in our research. On the one hand, the sample size of our research was limited, which probably made the model estimates biased. On the other hand, the final model can only be applied to the critically ill patients’ population who are not treated with CRRT or ECMO and cannot be extrapolated. Finally, we considered only 5 covariates, including age, weight, ALB, creatinine clearance, and APACHE-Ⅱ score, likely ignoring other potentially influential factors in the clinic. Therefore, the future work will expand the sample size and the influencing factors for further research.This study established a polymyxin B population PK model for critically ill patients. Since age and ALB levels were discovered as two key concomitant variables, an outstanding contribution was made in directing the individualized dosing of polymyxin B in critically ill patients. The increment of ALB in patients enhanced the possibility of getting the best exposure, while the possibility of obtaining an AUC/MIC ≥ 50 decreased with older age. In order to achieve therapeutic efficacy, the dose of polymyxin B needs to be heightened for critically ill elderly patients who often have lower ALB levels.


Androgens are closely associated with functions of hippocampal learning, memory, and synaptic plasticity. The zinc transporter ZIP9 (SLC39A9) regulates androgen effects as a binding site distinct from the androgen receptor (AR). However, it is still unclear whether androgens regulate their functions in hippocampus of mice through ZIP9. Compared with wild-type (WT) male mice, we found that AR-deficient male testicular feminization mutation (Tfm) mice with low androgen levels had learning and memory impairment, decreased expression of hippocampal synaptic proteins PSD95, drebrin, SYP, and dendritic spine density. Dihydrotestosterone (DHT) supplementation significantly improved these conditions in Tfm male mice, although the beneficial effects disappeared after hippocampal ZIP9 knockdown. To explore the underlying mechanism, we first detected the phosphorylation of ERK1/2 and eIF4E in the hippocampus and found that it was lower in Tfm male mice than in WT male mice, it upregulated with DHT supplementation, and it downregulated after hippocampal ZIP9 knockdown. Next, we found that the expression of PSD95, p-ERK1/2, and p-eIF4E increased in DHT-treated mouse hippocampal neuron HT22 cells, and ZIP9 knockdown or overexpression inhibited or further enhanced these effects. Using the ERK1/2 specific inhibitor SCH772984 and eIF4E specific inhibitor eFT508, we found that DHT activated ERK1/2 through ZIP9, resulting in eIF4E phosphorylation, thus promoting PSD95 protein expression in HT22 cells. Finally, we found that ZIP9 mediated the effects of DHT on the expression of synaptic proteins PSD95, drebrin, SYP, and dendritic spine density in the hippocampus of APP/PS1 mice through the ERK1/2-eIF4E pathway and affected learning and memory. This study demonstrated that androgen affected learning and memory in mice through ZIP9, providing new experimental evidence for improvement in learning and memory in Alzheimer’s disease with androgen supplementation. Androgens are steroid hormones synthesized in the gonads, adrenal glands, and brain  and are Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive memory impairment and cognitive impairment , with pa2+. It is also the only ZIP protein that can couple with G-proteins during signal transduction across membranes (Testicular feminization mutation (Tfm) mice are androgen receptor (AR)-deficient  and haveembranes , 20. Recembranes , migratiembranes  and bladembranes  cancer cembranes , 25, andembranes  and rat embranes  Sertoli embranes . HoweverIn this study, we explored whether androgens induced by ZIP9 affected learning, memory and hippocampal synaptic plasticity in Tfm male mice and investigated the underlying mechanism. Further, we examined whether learning and memory of AD animal model-APP/PS1 mice was mediated through this mechanism.Female Tfm and male C57BL/6J mice were purchased from Jackson Laboratory  and Vital River Laboratory Animal Technology Co., Ltd  respectively. We induced mating of Tfm female with male C57BL/6J mice which produced the following offspring types: wild-type (WT) female, WT male, Tfm female, and Tfm male. All offspring were genotyped using real-time polymerase chain reaction (PCR), and only WT and Tfm male mice were selected for this study. APP/PS1 mice were provided by the Vital River Laboratory Animal Technology Co., Ltd. . The mice were raised and bred at the Experimental Animal Research and Service Center of Hebei Medical University under conditions of constant temperature (22 ± 2°C), constant humidity (55 ± 5%), lighting (12-h light/dark cycle), and free access to food and water. All animal experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by Laboratory Animal Ethical and Welfare Committee of Hebei Medical University.2) in a humidified atmosphere. After digestion with 0.25% trypsin at 85%-90% confluency, the cells were seeded into a 6-well plate, and virus infection experiments were carried out to establish ZIP9 knockdown or overexpression HT22 stable cell lines. Subsequently, the cells were transferred into 6 or 24-well plates for Western blot and immunofluorescence staining. With or without pretreatment with 100 nM ERK1/2 inhibitor SCH772984  or 25 nM eIF4E inhibitor eFT508  for 2 h, the cells in the experimental groups were treated with 10 nM dihydrotestosterone  for 24 h, and those in the control group were treated with an equal volume of dimethyl sulfoxide (DMSO).Mouse hippocampal neuron HT22 cells were cultured in phenol red-free DMEM/F12 medium  containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under conditions  and the corresponding negative control lentivirus were provided by Genechem Inc. . HT22 cells were infected with the lentivirus for 12 h at 20-30% confluency. The HT22 cells were then cultured with fresh medium for 72 h until 4.5µg/mL puromycin was added for 48 h to kill uninfected cells. Stable clones were selected using 2.25 µg/mL puromycin after cell passaging, and stable ZIP9 knockdown or overexpression HT22 cell lines were established.HT22 cells were fixed with 4% paraformaldehyde (PFA) for 15 min, blocked with 10% donkey serum for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-PSD95 , rabbit anti-phospho-ERK1/2 , and rabbit anti-phospho-eIF4E . The next day, the cells were incubated with donkey anti-rabbit fluorescent secondary antibody  for 2 h at room temperature in the dark. They were then counterstained with 4’,6-diamidino-2-phenylindole (DAPI)  for 10 min and sealed with anti-fluorescence quenching sealing tablets . Images were taken using a laser confocal microscope , and the average optical density was analyzed using Fiji software . Intra and inter-assay coefficients of variation were 2.44%-4.95% and 4.07%-9.81% respectively.After anesthetizing 6-month-old APP/PS1 mice with isoflurane , small incisions were made in the scrotums to remove the testes in the experimental group, while the scrotums were cut open and sutured without hurting the testes in the sham operation group.13 v.g./ml) of virus was injected into the bilateral CA1 region.Adeno-associated virus (AAV9) and negative control virus with GV478 as a vector were provided by Genechem Inc. . The mice were anesthetized with isoflurane and fixed on a brain stereotaxic instrument  lying prone. After the anterior and posterior fontanelle were fully exposed, the CA1 region  was located by the mouse brain atlas . Through the microinjection system, 1μL , distance (%) in the novel arm, and the number of entries into the novel arm.A 3-day new object recognition experiment was performed in an open field box measuring 50×50×40 cm. On day 1, the mice were placed in a box for 5 min of adaptive training with free exploration, facing the sidewall close to the experimenter. On day 2, two identical objects were placed in the left and right corners away from the experimenter and 10 cm from each sidewall. Mice were placed in the box with their backs to the objects and allowed to explore freely for 10 min. On day 3, one of the original objects was replaced with a novel object of different color and shape, but having the same volume. The exploration time of the mice for the two objects was recorded within 10 min, and the exploration distance was 2-3 cm from the objects. Exploration behavior included placing the front paw on the object, smelling the object, licking the object. Holding a pose or climbing an object without moving is not an exploration of that object. The discrimination index represented by the ratio of exploration time of the novel object to the total exploration time, was calculated.The Morris water maze test was used to detect spatial learning and memory in the mice. The circular pool (diameter 120 cm) was divided into four quadrants, and the platform  was placed in any quadrant 1 cm underwater. In the orientation navigation trial, the mice were placed into the water facing the pool wall from four quadrants, and the time to find the platform was within 60 s, that is, the escape latency. If they could not find the platform in 60 s, the mice were guided to the platform and remained there for 15 s, and the escape latency was recorded as 60 s. The distance before finding the platform and swimming trajectories were recorded. On the 6th day, a spatial probe trial was performed. After the platform was removed, mice were placed in water. The number of mice crossing the position of the platform within 60s and the time spent in the target quadrant were recorded.Radioimmunoprecipitation assay lysate containing phenylmethylsulfonyl fluoride and phosphatase inhibitors was added to the samples, and proteins were extracted for quantitative analysis. After denaturation, 25 μg of protein was loaded onto a 10% sodium dodecyl sulphatepolyacrylamide gel for protein separation and electrotransferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-PSD95 , rabbit anti-phospho-eIF4E , mouse anti-eIF4E  , rabbit anti-GAPDH , rabbit anti-phospho-ERK1/2 , mouse anti-Erk1/2 , rabbit anti-ZIP9 antibody , rabbit anti-synaptophysin , and rabbit anti-drebrin . The membranes were then incubated with goat anti-rabbit fluorescent secondary antibody  or goat anti-mouse secondary antibody  in dark incubation boxes for 2 h. Finally, an Odyssey imaging system  was used for visualization and analysis. The relative expression of the target protein was calculated according to the gray value of β-actin or GAPDH as a reference, and the phosphorylation level of the proteins was determined by the ratio of phosphorylated proteins to total proteins. Intra and inter-assay coefficients of variation were 1.86% to 4.73% and 4.28% to 9.77% respectively.After the behavioral study, mice were deeply anesthetized with isoflurane, perfused with PBS, and fixed with 4% PFA. Their brains were removed and fixed in 4% PFA for 24 h. Brains were cut from the superior colliculus to the optic chiasma and separated along the median sagittal plane. The left parts were prepared for Golgi staining, and the right parts were routinely dehydrated, waxed, embedded, and cut into 5 μm-thick sections. After dewaxing, hydration, high pressure antigen repair, and blocking, the sections were incubated at 4 °C overnight with the following primary antibodies: rabbit anti-PSD95 , rabbit anti-synaptophysin , rabbit anti-drebrin . Subsequently, the sections were incubated with goat anti-rabbit IgG polymer labeled with biotin  for 30 min, horseradish enzyme-labeled streptomycin for 1 h, and DAB staining. The hippocampal CA1 region was observed and imaged under a 40× light microscope , and the average optical density was analyzed using Fiji software . Intra-assay coefficients of variation were 2.03% to 4.89%.Golgi staining was performed according to the protocol provided in the Golgi staining kit . After 24 h of post-fixation, the left brain was immersed in mordant (Reagent A and Reagent B were mixed at 1:1) and stained for 14 days at room temperature in the dark. They were then placed in 30% sucrose solution, dehydrated at 4 °C for 48 h, and cut into 100-μm sections with oscillating tissue slicers. The sections were incubated with staining solution at room temperature for 30 min, followed by incubation with chromogenic solution for 20 min at room temperature in the dark. They were then dehydrated, made transparent, and sealed with neutral resin. The secondary or tertiary dendritic spines of apical dendrites in the hippocampal CA1 region were observed and imaged under a 100× light microscope . Dendritic spine density was analyzed using Fiji software. Three sections were selected from each mouse and three neurons were selected from each section. Intra-assay coefficients of variation were 2.08% to 4.55%.P > 0.1). Levene’s test for homogeneity of variance was conducted on data from multiple groups. One-way analysis of variance (ANOVA) was performed for data with a normal distribution (P > 0.1) and homogeneity of variance (P > 0.1), and post hoc multiple comparisons were performed using the LSD test. The Kruskal-Wallis H test was used to compare multiple groups of quantitative data with non-normal distribution (P < 0.1), and post hoc multiple comparisons were performed with the independent-samples Kruskal-Wallis Test. The significant differences in escape latency at 1-5 days were assessed using two-way repeated-measures ANOVA. Differences were considered significant at P < 0.05. To ensure validity, all the data was analyzed post-hoc with G*Power (www.gpower.hhu.de), and the statistical power was set at equal to or greater than to 0.8.SPSS26.0 statistical software was used for analysis. The results are expressed as mean ± standard deviation (SD). The Shapiro-Wilk test for normality was performed, and the Student’s t-test was used for two-sample comparisons of normally distributed data (t(10) = -0.084, P = 0.935, Cohen’s d = 0.05) . To determine whether androgen affects the learning and memory of Tfm male mice through ZIP9, we transfected hippocampal neurons with AAV9-ZIP9-RNAi and control virus (AAV9-nc-RNAi). The expression of hippocampal ZIP9 in the Tfm+ZIP9-RNAi group was significantly lower than that in the Tfm+nc-RNAi group (t(10) = 6.746, P < 0.05, Cohen’s d = 3.89) . We tested the effect of DHT induced by ZIP9 on the behavior of WT and Tfm male mice  = 5.059, P < 0.05, η2 = 0.297), the percentage of distance in the novel arm  = 5.790, P < 0.05, η2 = 0.325), and the number of entries into the novel arm  = 3.614, P < 0.05, η2 = 0.231) among all groups. The percentage of time spent in the novel arm, the percentage of distance in the novel arm, and the number of entries into the novel arm of the Tfm+nc-RNAi group were significantly lower than those of the WT+nc-RNAi and Tfm+nc-RNAi+DHT groups, whereas those of the Tfm+nc-RNAi+DHT group were higher than those of the Tfm+ZIP9-RNAi+DHT group .The results of the YM showed that there were significant differences in the percentage of time spent in the novel arm  = 12.547, P < 0.05, η2 = 0.511). The DI in the Tfm+nc-RNAi group was significantly lower than that in the WT+nc-RNAi group. DHT supplementation increased the DI of Tfm male mice, whereas the increase induced by DHT disappeared after hippocampal ZIP9 knockdown .The NOR test showed significant differences in discrimination index (DI) among all groups  = 3.502, P < 0.05, η2 = 0.203) and the distance to the target on the 5th day  = 4.657, P < 0.05, η2 = 0.280). The values of above parameters for the Tfm+nc-RNAi group were significantly higher than those for the WT+nc-RNAi and the Tfm+nc-RNAi+DHT groups, whereas the values for Tfm+nc-RNAi+DHT group were lower than those for the Tfm+ZIP9-RNAi+DHT group. The subsequent spatial probe trial revealed differences in the number of target crossings  and time in the target zone  = 7.598, P < 0.05, η2 = 0.388). The number of target crossings and time in the target zone of the Tfm+nc-RNAi group were lower than those of the WT+ncRNAi and Tfm+nc-RNAi+DHT groups, whereas those of the Tfm+nc-RNAi+DHT group were higher than those of the Tfm+ZIP9-RNAi+DHT group  = 17.942, P < 0.05, η2 = 0.729), drebrin  = 77.031, P < 0.05, η2 = 0.920), and SYP  = 16.593, P < 0.05, η2 = 0.713). Compared to the WT+nc-RNAi group, the optical density of PSD95, drebrin, and SYP in the Tfm+nc-RNAi group decreased significantly. DHT supplementation increased the optical density of these hippocampal proteins in Tfm male mice, while the increase induced by DHT disappeared after hippocampal ZIP9 knockdown  = 5.833, P < 0.05, η2 = 0.467), drebrin  = 7.278, P < 0.05, η2 = 0.552), and SYP  = 12.693, P < 0.05, η2 = 0.656), as observed by IHC staining  = 33.942, P < 0.05, η2 = 0.836). Compared to the WT+nc-RNAi group, the dendritic spine density in the hippocampi of the Tfm+nc-RNAi group decreased significantly. DHT supplementation increased the density of dendritic spines in Tfm male mice, but this increase was not observed after ZIP9 knockdown in the hippocampus .Golgi staining results showed significant inter-group differences in the density of dendritic spines  = 143.584, P < 0.05, η2 = 0.964) and eIF4E  = 31.446, P < 0.05, η2 = 0.855). Compared to the WT+nc-RNAi group, the phosphorylation of ERK1/2 and eIF4E in the Tfm+nc-RNAi group decreased significantly. DHT supplementation increased the phosphorylation of ERK1/2 and eIF4E in Tfm male mice, but this increase was not observed after ZIP9 knockdown in the hippocampus  = 17.814, P < 0.05, Cohen’s d = 25.193) ; the expression of PSD95  = 550.836, P < 0.05, η2 = 0.989), phosphorylation of ERK1/2  = 402.259, P < 0.05, η2 = 0.985), and eIF4E  = 14.350, P < 0.05, η2 = 0.705) in the nc-shRNA+DHT group were significantly higher than those in the nc-shRNA and ZIP9-shRNA+DHT groups  = 39.748, P < 0.05, η2 = 0.869), p-ERK1/2  = 17.002, P < 0.05, η2 = 0.739), and p-eIF4E  = 33.090, P < 0.05, η2 = 0.847) in the nc-shRNA+DHT group were significantly higher than those in nc-shRNA and the ZIP9-shRNA+DHT groups  = -6.160, P < 0.05, Cohen’s d = -8.711, ); expression of PSD95  = 1003.038, P < 0.05, η2 = 0.994), phosphorylation of ERK1/2  = 385.322, P < 0.05, η2 = 0.985), and eIF4E  = 291.648, P < 0.05, η2 = 0.980) in the nc-oe+DHT group were significantly higher than those in the nc-oe group, but lower than those in the ZIP9-oe+DHT group  = 126.544, P < 0.05, η2 = 0.955), p-ERK1/2  = 132.102, P < 0.05, η2 = 0.957) and p-eIF4E  = 86.879, P < 0.05, η2 = 0.935) in the nc-oe+DHT group were significantly higher than those in the nc-oe group, but lower than those in the ZIP9-oe+DHT group  = 374.158, P < 0.05, η2 = 0.986), eIF4E  = 124.188, P < 0.05, η2 = 0.959), and the expression of PSD95  = 274.867, P < 0.05, η2 = 0.981). The phosphorylation of ERK1/2 and eIF4E and the expression of PSD95 in the nc-oe+DHT group were higher than those in the nc-oe group and lower than those in the ZIP9-oe+DHT group, whereas these values for the ZIP9-oe+S+DHT group were lower than those for the ZIP9-oe+DHT group  = 59.122, P < 0.05, η2 = 0.917), p-eIF4E  = 55.533, P < 0.05, η2 = 0.912), and PSD95  = 48.888, P < 0.05, η2 = 0.902) were significantly different in all groups. The optical densities of p-ERK1/2, p-eIF4E, and PSD95 in the nc-oe+DHT group were higher than those in the nc-oe group and lower than those in the ZIP9-oe+DHT group, whereas those in the ZIP9-oe+S+DHT group were lower than those in the ZIP9-oe+DHT group  = 320.397, P < 0.05, η2 = 0.984) and the expression of PSD95  = 497.501, P < 0.05, η2 = 0.989). The phosphorylation of eIF4E and the expression of PSD95 in the nc-oe+DHT group were higher than those in the nc-oe group and lower than those in the ZIP9-oe+DHT group, whereas those in the ZIP9-oe+eFT+DHT group were lower than those in the ZIP9-oe+DHT group  = 74.668, P < 0.05, η2 = 0.933) and PSD95  = 59.422, P < 0.05, η2 = 0.918. The optical densities of p-eIF4E and PSD95 in the nc-oe+DHT group were higher than those in the nc-oe group and lower than those in the ZIP9-oe+DHT group, whereas these values for the ZIP9-oe+eFT+DHT group were lower than those for the ZIP9-oe+DHT group  = 13.241, P < 0.05, η2 = 0.525), the percentage of distance travelled  = 7.551, P < 0.05, η2 = 0.386), and the number of entries  = 7.675, P < 0.05, η2 = 0.390) in the ovel arm. The values of above parameters for Cast+nc-RNAi group were significantly lower than those for the sham+nc-RNAi and Cast+nc-RNAi+DHT groups, whereas the values for Cast+nc-RNAi+DHT group were higher than those for the Cast+ZIP9-RNAi+DHT group  = 11.338, P < 0.05, η2 = 0.486). The DI of the Cast+nc-RNAi group was significantly lower than that of the sham+nc-RNAi group. DHT supplementation increased the DI of castrated APP/PS1 mice, while this increase disappeared after ZIP9 knockdown in the hippocampus  = 4.307, P < 0.05, η2 = 0.264) and the distance to the target on the 5th day  = 7.271, P < 0.05, η2 = 0.377). The values of above parameters for the Cast+nc-RNAi group were significantly higher than those of the Sham+nc-RNAi and Cast+nc-RNAi+DHT groups, whereas the values for the Cast+nc-RNAi+DHT group were lower than those for the Cast+ZIP9-RNAi+DHT group. The subsequent spatial probe trial demonstrated significant differences in the number of target crossings  = 3.803, P < 0.05, η2 = 0.241) and time in the target zone  = 9.821, P < 0.05, η2 = 0.450). The number of target crossings and time in the target zone of the Cast+nc-RNAi group were lower than those of the sham+nc-RNAi and Cast+nc-RNAi+DHT groups; however, those of the Cast+nc-RNAi+DHT group were lower than those of the Cast+ZIP9-RNAi+DHT group  = 10.479, P < 0.05, η2 = 0.611), drebrin  = 12.782, P < 0.05, η2 = 0.657) and SYP  = 30.519, P < 0.05, η2 = 0.821). Compared with the Sham+nc-RNAi group, the optical density of PSD95, drebrin, and SYP in the Cast+nc-RNAi group decreased significantly. The DHT supplementation increased the optical density of these proteins in castrated APP/PS1 mice, while the increase vanished after ZIP9 knockdown in the hippocampi  = 27.055, P < 0.05, η2 = 0.802) and drebrin  = 9.642, P < 0.05, η2 = 0.591) and SYP  = 6.262, P < 0.05, η2 = 0. 484) in the hippocampi as observed by IHC staining  = 30.471, P < 0.05, η2 = 0.820). Compared to the Sham+nc-RNAi group, the dendritic spine density in the hippocampi of the Cast+nc-RNAi group decreased significantly. DHT supplementation increased dendritic spine density in castrated APP/PS1 mice, but this increase was not observed after ZIP9 knockdown in the hippocampus .Golgi staining results showed a significant inter group differences in the density of dendritic spines  = 31.214, P < 0.05, η2 = 824) and eIF4E  = 13.242, P < 0.05, η2 = 0. 665). Compared to the Sham+nc-RNAi group, the phosphorylation of ERK1/2 and eIF4E in the Cast+nc-RNAi group decreased significantly. DHT supplementation improved the phosphorylation of ERK1/2 and eIF4E in castrated APP/PS1 mice, while improvement was not observed after ZIP9 knockdown in the hippocampus  may be a key downstream signal molecule of ZIP9-mediated androgen effect. Profaska-Szymik et al.  found th studies , 47–51 h studies –55. The  studies . The cap studies , 58. Gin studies  found upAlthough we found that DHT promoted the phosphorylation of ERK1/2, eIF4E and the expression of PSD95 through ZIP9, the mechanism still required experimental verification. We found that pretreatment with SCH772984, a specific inhibitor of ERK1/2, significantly inhibited the phosphorylation of ERK1/2, eIF4E and expression of PSD95. Pretreatment with eFT508, a specific inhibitor of eIF4E, significantly inhibited the phosphorylation of eIF4E and expression of PSD95 in ZIP9-overexpression HT22 cells. These results are consistent with those of other studies showing that ERK1/2 activation can regulate synaptic protein synthesis by phosphorylating eIF4E, which subsequently affects synaptic plasticity , 61. Comin vivo leads to cognitive decline, which was measured by learning and memory (The serum level of total or free testosterone in AD patients is significantly lower than that in normal elderly men , and thed memory . Studiesd memory . This sud memory –68. APP/d memory , 70. Obvd memory , 72. ThiThe ZIP9 has been described as a membrane androgen receptor , 73–75. However the findings of this study have to be seen in light of some limitations. Firstly, considering that the topic of this study is the effects of DHT mediated by ZIP9 on hippocampal synaptic plasticity and learning and memory, we did not detect hippocampal Aβ in APP/PS1 mice, although senile plaque formed by excessive deposition of Aβ is one of the pathological features of AD. Therefore, we are unable to know the effects of DHT mediated by ZIP9 on hippocampal pathology in APP/PS1 mice. Secondly, ZIP9 has both androgen signaling and zinc transport functions, and the mechanism of zinc ion on learning and memory is still unclear. Since this study focused on ZIP9-mediated androgen influence on synaptic plasticity and learning memory in mice, its zinc transport function was not investigated. Finally, only male mice were used in this study, and the results of this study may not be applicable to female animals because of gender differences.Based on the above experiments, we concluded that ZIP9 mediated the effects of DHT on improving hippocampal synaptic plasticity-related proteins and dendritic spine density in Tfm male mice through the ERK1/2-eIF4E pathway, improving learning and memory. DHT can also affect learning and memory in castrated APP/PS1 mice through this mechanism. This study provides important experimental data for further research into the use of androgen supplementation in Alzheimer’s Disease to improve learning and memory.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Hebei Medical University Laboratory Animal Welfare and Ethics Committee.HXC and SL designed the research, writing-review, editing, project administration. LS and HC performed the experimental phase, methodology, data curation, writing-original draft. DQ, BZ, FG, YZ and CW contributed toward investigation, and visualization. All authors contributed to the article and approved the submitted version.


Plagiothecium deposited in the herbarium of Pakistan Museum of Natural History (PMNH) collected during a Japanese lead project on Cryptogams in the Western Himalaya (Pakistan) shows that the material consists of five taxa. Of the studied samples, the most common taxa were from the P.denticulatum complex, including Plagiotheciumdenticulatumvar.obtusifolium, new to Pakistan. Examination of the rest of the collection showed that it consists of specimens with a unique combination of qualitative and quantitative characteristics of their gametophyte. For example, for small plants, with small asymmetrical, folded leaves, gradually tapering into long, acuminate, not denticulate apex, whose leaf cells are long and narrow, making the cell areolation tight, the name Plagiotheciumfilifolium is proposed. For other plants with large leaves, loosely arranged on the stem, concave, symmetrical to slightly asymmetrical, with denticulate apex and long decurrency composed of rectangular and spherical, inflated cells, the name Plagiotheciumhiguchii is proposed. However, within this material, specimens differ in terms of the length and width of the leaf cells and therefore, within this taxon, two varieties are distinguished: Plagiotheciumhiguchiivar.higuchii and Plagiotheciumhiguchiivar.brevicellum.A revision of specimens of  The northern part of Pakistan has a suitable climate and ambience for bryophytes, while the southern part of this country is nearly unexplored because it is far drier, having hot weather and some parts with arid and desert ecosystems. Thereby, due to difficult fieldwork, geographic and climatic conditions, this country is generally still poorly studied bryologically but mainly due to the lack of a resident bryologist. The scattered literature principally covers northern Pakistan and Western Himalaya .The first checklist of mosses of Pakistan based on previous literature, and herbarium specimens, from Punjab and Khyber Pakhtunkhwa provinces was provided by Pottiaceae) consisting of 79 species  Schimp. and P.nemorale (Mitt.) A.Jaeger. Later, P.nemorale from Pakistan. Plagiotheciumcavifolium (Brid.) Z.Iwats. from the Indian-administrated area of Kashmir. Subsequent studies  we can see that the ely poor . Taking PMNH). This collection was made by Japanese bryologists during their Cryptogamic Expedition in Pakistan, which was organized through the collaboration of the National Science Museum , Tokyo, Japan (herbarium TNS) and the Pakistan Museum of Natural History (LOD), Poland.The current study is based exclusively on the Pakistani material, on the specimens deposited at the herbarium of Botanical Sciences Division, Pakistan Museum of Natural History , Galiyat-region, Swat Valley, Lower Dir (Khyber Pakhtunkhwa Province), and some selected areas of Azad Jammu and Kashmir (Pakistan).Measurements were made in accordance with the methodology proposed by Plagiotheciumhiguchiivar.higuchii and Plagiotheciumhiguchiivar.brevicellum – were statistically compared to see whether the differences are significant. However, due to the lack of normality in the distributions of individual variables, a non-parametric U-Mann-Whitney test was performed.The features of two similar specimens – later named Plagiotheciumdenticulatum complex. Thus, P.cavifolium, P.latebricola and P.nemorale, previously reported from this area, were not identified in the samples surveyed. The P.denticulatum complex dominated the examined material, within which the largest number of samples was represented by P.denticulatumvar.denticulatum, the remaining specimens belonged to a new taxon, not previously recorded from Pakistan, P.denticulatumvar.obtusifolium (Turner) Moore (M. Higuchi 20499).The conducted revision shows that the entire analyzed material is not very diverse. Most of the tested specimens belong to the Taxon classificationPlantaeHypnalesPlagiotheciaceae(Hedw.) Schimp.E16791D4-8D03-5792-A48F-D95B2F9B9542Plagiotheciumdenticulatum (Hedw.) Schimp., Bryologia Europaea 5: 190, 501, Tab. VIII (1851).≡HypnumdenticulatumLectotype (fide n.v.).  Hedw., Species Muscorum Frondosorum 237 (1801). ype fide : “St. CrPlagiotheciumdenticulatum has been recorded on soil and boulders.Plants green to yellowish green, with metallic luster; stem in cross section rounded, 200–250 μm in diameter; leaves not folded, julaceous, elliptical-ovate, asymmetrical; leaves 2.2–3.0 × 1.0–1.3 (M 2.6 × 1.1) mm, shortly tapering to acute, denticulate apex; costae two, extending usually to ½ of the leaf length, ranging 440–810 (M 625) μm; cell areolation loose; length and width of cells very variable, but dependent on location: 85–140 × 15–28 (M 113 × 22) μm at the apex; 120–210 × 15–30 (M 165 × 23) μm at midleaf; 116–205 × 18–37 (M 160 × 28) μm toward insertion; broad decurrency 400–700 (M 555) μm; alar cells rounded, clearly inflated, 45–90 × 20–50 (M 68 × 35) μm; sporophytes not found in this material  Moore27ED5B4D-F6F5-5939-939B-20157669594BPlagiotheciumdenticulatumvar.obtusifolium (Turner) Moore, Proceedings of the Royal Irish Academy 1: 424 (1873).≡Hypnumdenticulatumvar.obtusifolium Turner, Muscologiae Hibernicae Spicilegium 146, T. 12, f. 2 (1804). ≡PlagiotheciumobtusifoliumHolotype: fig. 2, tabela 12 “T. 12, f. 2” (Turner 1804: 237) (fig. 3). Epitype .  (Turner) J.J.Amann, Mémoire de la Société Vaudoise des Sciences Naturelles 3: 61 (1928). =PlagiotheciumsandbergiiLectotype ; isolectotypes: NY507114!, available online; US70396!, available online; FH220148. Additional original material from locus classicus (not signed “No. 1174”): 456 NY507115!, available online. Additional Sandberg material, potentially from locus 457 classicus: PC0132605! Additional Sandberg material: FH220147.  Renauld & Cardot, Contributions from the United States National Herbarium, 3: 274 (1895), =Plagiotheciumdenticulatumvar.auritumLectotype .  Kern, Jahresbericht der Schlesischen Gesellschaft für Vaterländische Cultur 91(Abt. 2b): 97 (1914). Plagiotheciumdenticulatumvar.obtusifolium (M. Higuchi 20499) has been recorded on soil.Plants light green, with metallic luster; stem in cross section rounded, 150–200 μm in diameter; the central strand well developed, epidermal cells 12–20 × 15–31 (M 16 × 23) μm, the parenchyma thin-walled, 25–43 × 21–39 (M 34 × 30) μm; leaves very concave, not folded, julaceous, elliptical-ovate, very slightly asymmetrical, 1.6–1.7 × 0.8–0.95 (M 1.65 × 0.87) mm; the apex obtusely-apiculate; costae two, extending usually up to 1/3 or ½ of the leaf length, ranging 250–530 (M 390) μm; cell areolation loose; length and width of cells very variable, but dependent on location: 85–140 × 15–18 (M 113 × 17) μm at the apex; 95–170 × 12–18 (M 133 × 15) μm at midleaf; 98–200 × 15–25 (M 150 × 20) μm toward insertion; broad decurrency 260–400 (M 330) μm; alar cells rounded-rectangular, 51–98 × 21–31 (M 75 × 26) μm; sporophytes not found in this material  were characterized by a unique combination of qualitative and quantitative features of their gametophytes. They did not match any of the taxa of the genus currently known in the Northern and Southern Hemispheres.The next two samples (M. Higuchi 20460) represents two morphotypes that are microscopically very different from each other. This material differs in the length and width of the leaf, the shape of the apex, the length and width of the cells of the top, middle and basal part of the leaf – which are one of the most important taxonomic features for the whole genus. In addition, the result of the U Mann-Whitney test showed that the length and width of the cells of individual parts of the leaf of these morphotypes differ statistically significantly (p<0.001) from each other. Thus, it was proposed to recognise two varieties – Plagiotheciumhiguchiivar.higuchii and P.higuchiivar.brevicellum.The specimen collected by M. Higuchi  has been recorded on soil.Plants green-yellow to golden-gold, without metallic luster; stems complanate-foliate, 3.0–4.0 cm long, in cross-section rounded, with a diameter of 290–360 (M 325) μm, the central strand well developed, epidermal cells 11–22 × 13–32 (M 16 × 22) μm, the parenchyma thin-walled, 26–50 × 23–45 (M 38 × 34) μm; leaves quite loosely arranged on the stem, concave, symmetrical to asymmetrical, ovate, those leaves from the middle of the stem 3.4–4.3 × 1.2–1.9 (M 3.8 × 1.5) mm; the apex acuminate and denticulate; costae two, thick and strong, usually to 1/2 of the leaf length, reaching 0.5–1.2 mm; laminal cells symmetrical, in unregulated transverse rows, the length and width very variable, but dependent on location: 107–250 × 16–24 (M 178 × 20) μm at apex, 139–266 × 21–33 (M 203 × 27) μm at mid-leaf and 139–266 × 21–33 (M 203 × 27) μm towards insertion, cell areolation very loose; decurrency long, 300–500 (M 400) μm, composed of 4–6 rows of rectangular and spherically inflated cells, 48–144 × 26–70 (M 96 × 48) μm. Sporophytes so far unknown  chosen here as the holotype of Plagiotheciumhiguchii.The present species is named in honor of Professor Masanobu Higuchi, who participated in the Studies on Cryptogams in the Western Himalayas in Pakistan project, and who collected the specimen  has been recorded on soil.Plants green-yellow to golden-gold, without metallic luster. Stems complanate-foliate, 3.0–4.0 cm long, in cross-section rounded, with a diameter of 310–388 μm, epidermal cells 10–16 × 21–33 (M 13 × 27) μm, the parenchyma thin-walled, 14–58 × 18–51 (M 34 × 35) μm; leaves quite loosely arranged on the stem, concave, symmetrical to gently asymmetrical, ovate, those leaves from the middle of the stem 2.9–3.2 × 1.0–1.2 (M 3.1 × 1.1) mm; the apex acute and apiculate, denticulate; costae two, thick and strong, usually to 1/2 of the leaf length, reaching 0.5–1.5 (M 1.0) mm; laminal cells symmetrical to slightly asymmetrical, the length and width very variable, but dependent on location: 70–170 × 10–19 (M 120 × 15) μm at apex, 100–200 × 14–26 (M 150 × 20) μm at mid-leaf and 100–200 × 19–36 (M 150 × 28) μm towards insertion, cell areolation loose; decurrency long, 390–650 (M 520) μm, composed of 4–6 rows of rectangular and spherically inflated cells, 36–116 × 18–67 (M 76 × 43) μm. Sporophytes unknown so far  has been recorded on boulders.Plants green-yellow, without metallic luster; stems complanate-foliate, 1.0–1.5 cm long; cross-section rounded, with a diameter of 200–300 μm, the central strand well developed, epidermal cells 9–13 × 11–15 (M 11 × 13) μm, the parenchyma thin-walled, 15–21 × 16–28 (M 18 × 22) μm; leaves folded, most strongly in the upper part of leaves, concave, asymmetrical, those leaves from the middle of the stem 1.6–2.2 × 0.5–0.8 (M 1.9 × 0.66) mm; leaves gradually tapering to long, acuminate, not denticulate apex; costae two, very thin and delicate, extending up to 1/2 of the leaf length, reaching 140–423 (M 282) μm; laminal cells rather symmetrical, the length and width very variable, but dependent on location: 97–160 × 7–10 (M 127 × 8) μm at apex, 105–180 × 7–9 (M 150 × 8) μm at mid-leaf and 70–165 × 8–11 (M 118 × 9) μm towards insertion, cell areolation tight; decurrency short, 220–530 μm, composed of 3 rows of gently inflated, rectangular cells, 47–147 × 12–21 (M 97 × 16) μm. Sporophytes unknown so far  Bruch and Schimp., but this is now considered to be a synonym of P.nemorale  were considered a very low number (Plagiothecium recorded from China (20 taxa)  Müll. Hal. (Vesiculariasubulata (Broth.) J.T. Wynns & Frank Müll. The aforementioned can be characterized as medium-sized plants, complanate or fluffy, leaves weakly overlapping, flat, short, broad, ovate, asymmetric, with erect margins and gemmae produced in dense clusters at leaf tips    Fig. .Plagiotheciumfilifolium has been recorded so far from: Gilgit-Baltistan, Nanga Parbat, Mazeno Base Camp, at 4000 m (M. Higuchi 20479)  Fig. .Plagiotheciumhiguchii var.higuchii has been recorded so far from: Gilgit-Baltistan, Nanga Parbat, Mazeno Base Camp, at 4000 m (M. Higuchi 20460)  PlagiothePlagiotheciumnemorale has been recorded so far from: Gilgit-Baltistan, Minimarg, Burzil Pass, 9000 to 10 000 m and 10 000 to 11 000 m (P.sylvaticum); Khyber Pakhtunkhwa, Mansehra, Sharan Forest, 2400 m Plagiothe


The combinational DOG/MOFA/ICG nanoparticles with near-infrared laser irradiation showed better anti-tumor efficacy than individual chemotherapy or photothermal therapy, with very low hemolysis and inappreciable toxicity for L929 cells. This co-self-assembly of the ICG and the chemotherapy drug (GEM) provides a novel tactic for the rational design of multifunctional nanosystems for targeting drug delivery and theranostics.The carrier-free chemo-photothermal therapy has become a promising strategy to improve anti-cancer therapeutic efficacy owing to the combination of chemotherapy and photothermal therapy, with improved chemotherapy drug pharmacodynamics and pharmacokinetics, high drug loading, and reduced toxicity. We designed a novel carrier-free targeting nanoparticles, co-self-assembled amphiphilic prodrugs 3′,5′-dioleoyl gemcitabine (DOG), and tumor-targeted γ-octadecyl folate (MOFA), with encapsulated US Food and Drug Administration (FDA)-approved photosensitizer indocyanine green (ICG) for synergistic chemo-photothermal therapy. The DOG linking oleic acid to the sugar moiety of gemcitabine (GEM) showed better self-assembly ability among GEM amphiphilic prodrugs linking different fatty acids. The readily available and highly reproducible 3′,5′-dioleoyl gemcitabine/γ-octadecyl folate/indocyanine green (DOG/MOFA/ICG) nanoparticles were prepared by reprecipitation and showed nano-scale structure with mono-dispersity, great encapsulation efficiency of ICG (approximately 74%), acid- and laser irradiation-triggered GEM release  Cancer-related deaths are becoming a major global health problem . Lung caUntil recently, co-delivery of multifunctional drugs has been made in various nano-sized carriers for encapsulating multiple agents and delivering them to cancer cells . These nHere, we developed carrier-free functionalized nanoparticles (DOG/MOFA/ICG nanoparticles) for chemo-photothermal therapy to improve the pharmacokinetics of GEM and enhance cell lethality. As shown in 1H NMR spectra were recorded on a 400 MHz instrument  with TMS as an internal standard. MS experiments were performed on a micro TOF-Q II , employing electrospray ionization in positive mode.All materials, solvents, and intermediates were purchased from commercial suppliers and were used without further purification. GEM, ICG, folate , oleic acid, linoleic acid, myristic acid, decanoic acid, heptanoic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1-octadecanol and pyrene were purchased from Leyan . Cell Counting Kit-8 (CCK-8), Annexin V-FITC Apoptosis Detection Kit, 4′,6-diamino-2-phenylindole (DAPI) were purchased from Beyotime Bio-Technology Co., Ltd. . Fetal bovine serum (FBS) was purchased from Gibco . Dulbecco’s Modified Eagle Medium (DMEM), trypsin, penicillin/streptomycin solution were obtained from Hyclone . A549 cancer cells  and L929 normal cells (a mouse fibroblasts cell line) were purchased from the American Type Culture Collection . In vivo studies were approved by the Institutional Animal Care and Use Committee (no. 2023-011) at Chengdu Medical College.Kunming mice  were purchased from Chengdu Dashuo Biological Institute . All animal experiments complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China. 2SO4. The organic layer was evaporated to get the residue which was purified by column chromatography using petroleum ether: ethyl acetate (5:1) to get pure products.#1): GEM was reacted with oleic acid.3′,5′-Dioleoyl gemcitabine : GEM was reacted with linoleic acid.3′,5′-Dilinoleoyl gemcitabine : GEM was reacted with myristic acid.3′,5′-Dimyristoyl gemcitabine : GEM was reacted with decanoic acid.3′,5′-Didecanoyl gemcitabine : GEM was reacted with heptanoic acid.3′,5′-Diheptanoyl gemcitabine : FA  was dissolved in 15 mL of N, N-dimethylformamide (DMF), 1-octadecanol , EDCI  and DMAP  were added. After stirring at room temperature for 48 h under argon, the reaction mixture was poured into 60 mL of acetone and stirred for 30 min to afford precipitate, which was filtrated, washed with acetone, and dried under vacuum.The different self-assembled nanoparticles were prepared by reprecipitation-dialysis method . BrieflyNext, a series of characterizations were implemented on the obtained DOG, DLG, DHG, DDG, and DMG nanoparticles. The zeta potential, size distribution, and polydispersity index (PDI) of obtained different nanoparticles were measured by laser particle size analyzer .DOG, DOG/MOFA, DOG/ICG, and DOG/MOFA/ICG nanoparticles were prepared according to the above screening results. Typically, DOG (24.0 mg); DOG (24.0 mg), and MOFA (2.4 mg); DOG (24.0 mg) and ICG (2.4 mg); DOG (24.0 mg), MOFA (2.4 mg) and ICG (2.4 mg) were respectively dissolved in 4 mL of DMSO in a round-bottomed flask and kept stirring for 4 h. The above solutions were slowly added into 20 mL of ultra-pure water and vigorously stirred for another 2 h. The obtained solutions were transferred into a 3.5 kDa molecular weight cut-off dialysis bag and dialyzed in ultra-pure water for 24 h, and the water was replaced every 4 h.® 50 Plus UV-vis spectrophotometer . The fluorescence spectrum was implemented on the RF-6000 Spectro fluorometer .Next, a series of characterizations on the obtained DOG, DOG/MOFA, DOG/ICG, and DOG/MOFA/ICG nanoparticles were performed. The zeta potential, size distribution, and PDI of obtained nanoparticles were analyzed by laser particle size analyzer. Transmission electron microscopy (TEM) was conducted on an HT7700 transmission electron microscope . The UV data of different nanoparticles was measured by a SpecordThe assembly mechanisms of DOG/MOFA/ICG nanoparticles were investigated using computer simulations. Molecular dynamics (MD) calculations were carried out using the Discovery Studio software based on the CharMM (Chemistry at Harvard Macromolecular Mechanics). In the simulation phase, the dielectric constant was 80 (simulating an aqueous environment), using a leapfrog Verlet dynamics integrator. In the first step, the energy minimization was performed using the steepest descent method . In the heating phase we simulated 50 ps (time step: 2 ps) at a target temperature of 300 K. The equilibrium phase was run for 100 ps to keep the temperature at 300 K. In the production step, 1,000,000 ps were run and the results were saved in 1,000 ps intervals (snapshots). The Fourier Transform infrared (FT-IR) spectrum was performed on a Thermo Fisher Nicolet Is5 infrared spectrometer .® 50 Plus UV-vis spectrophotometer. Methanol was added into 1 mL of self-assembled nanoparticles for ultrasonic emulsification. The absorbance was determined at 784 nm (ICG), with blank correction. A summary method for calculating drug loading (DL) and entrapment efficiency (EE) was shown as follows.The quantitative analysis of GEM, DOG, and ICG was obtained by establishing a standard curve through SpecordGEM = 320 nm, λICG = 784 nm). In addition, to investigate the influence of the laser irradiation on the release behavior of DOG/ICG and DOG/MOFA/ICG nanoparticles, in vitro drug release experiments were also implemented in the same way .The cumulative release of GEM from different nanoparticles was studied by the dialysis method to investigate the release behavior of different nanoparticles. At first, a total of 0.8 mL of different nanoparticle solutions were transferred to a dialysis bag (molecular weight cut-off = 3.5 kDa) and immersed in 30 mL PBS at pH 7.4, 6.5, and 5.5. The controlled release of the nanoparticles in the absence/presence of esterase enzyme was examined. The mixture solutions were gently shaken in a constant-temperature incubator . The dialysate (0.5 mL) in dialysis tubes was withdrawn at each predetermined time and replaced with fresh PBS with the same pH. Secondly, the release content of GEM and ICG was measured with the UV-vis spectrometer . For the stability of different nanoparticles in FBS, 900 μL different nanoparticles and 100 μL FBS were respectively mixed. Then, the PDI and size of nanoparticles in various mediums were examined for 7 days, respectively. Moreover, the stability of different nanoparticles under laser irradiation was studied, 1 mL of different nanoparticles were respectively transferred to EP tubes and irradiated by the 808 nm laser  with a power density of 1 W/cmex = 339 nm, λem = 350–500 nm) using an RF-6000 Spectro fluorometer . The intensity ratios of the first (371 nm) to the third (381 nm) peaks (I1/I3) were plotted with the copolymer concentration’s logarithm. The intersection of the two lines was the CMC value.The CMC of different nanoparticles was measured using a pyrene probe as a fluorescence probe . Briefly2 for 5 min. The temperature change was monitored by a Fluke TiS20 thermal infrared imaging camera .The 808 nm laser was exploited in the photothermal effect experiments. Ultra-pure water, free ICG (10 μg/mL), and different nanoparticles  were respectively transferred to EP tubes and irradiated by the 808 nm laser with 1 W/cmA fresh rabbit blood sample (2.0 mL) was stabilized by heparin sodium and then mixed with 4.0 mL of PBS (4°C). After centrifugation at 3,000 rpm for 5 min, the red blood cells (RBCs) were washed 3 times with cold PBS (4°C) by centrifugation-redispersion until the supernatant was clarified. Subsequently, the different nanoparticles were incubated with 2% (w/v) of RBCs dispersions at 37°C for 6 h with various concentrations. Further, PBS was used as a negative control (NC), and ultra-pure water was used as a positive control (PC). Finally, the samples were centrifugated at 4,000 rpm for 10 min, the supernatants were collected and the released hemoglobin was determined with a UV-vis spectrometer at 540 nm.(sample), A(negative), and A(positive) are the absorbance values of the sample group, the negative control group, and the positive control group, respectively.Where A2.A549 cancer cells (a human NSCLS cell line) and L929 normal cells (a mouse fibroblasts cell line) were all cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO5 cells per well in 0.5 mL DMEM and cultured for 24 h. Subsequently, 0.5 mL DMEM containing ICG, DOG/ICG, and DOG/MOFA/ICG nanoparticles with the same ICG concentration (10 μg/mL) was added to every well. The cells were further incubated at 37°C for 1 and 4 h, respectively. Thereafter, the cells were washed with cold PBS three times and fixed with 4% paraformaldehyde for 30 min at room temperature. After that, the cells were washed with cold PBS three times and stained with DAPI for 10 min. Finally, the slides were mounted and directly observed with an OLYMPUS BX63F fluorescence microscope. Meanwhile, the cell uptake was quantitatively analyzed by Novocyte Quanteon flow cytometry . The cells were collected, and washed with cold PBS three times. Finally, they were analyzed by flow cytometry.The experiments on cellular uptake were performed on fluorescence microscopy and flow cytometry. A549 cells were seeded in 24-well plates at 1 × 10To investigate the competitive inhibition of DOG/MOFA/ICG nanoparticle uptake via folate receptor-mediated endocytosis, the A549 cells were first incubated with free folate (1 mM) to block folate receptor binding and then further treated with DOG/MOFA/ICG nanoparticles for 4 h, then both attached and floating cells were collected, washed with cold PBS three times. Finally, they were analyzed by flow cytometry.4 cells/100 µL/well) were incubated in a 96-well plate 24 h prior to the experiments, and the different concentrations of GEM, free DOG, ICG, and different nanoparticles were given to the cell. In order to determine the synergistic chemo-photothermal therapy effect, A549 cells were given without and with near-infrared laser irradiation  for 5 min after 24 h of drug administration. After being incubated for another 24 h, cells were washed twice with PBS, added fresh culture medium (100 μL), along with 10 μL of CCK-8 solution, and further incubated for 4 h in the dark. The absorbance was read at 450 nm with a VICTOR Nivo multimode plate reader. The following A549 cells (1 × 10Dm)1 and (Dm)2 represent the IC50 values of treatments 1 and 2 applied separately, while D1 and D2 are the IC50 values of treatments 1 and 2 applied as a combination. for 48 h. A549 cells without the same treatment were used as a control. To measure the cell apoptosis quantitatively, floating and attached cells were also collected, washed with ice-cold PBS three times, and stained with Annexin V-FITC and propodium iodide (PI). Finally, they were analyzed by flow cytometry.A549 cells were seeded in 6-well plates at 3.0 × 10in vivo studies were approved by the Institutional Animal Care and Use Committee (no. 2023-011) at Chengdu Medical College. The mice were randomly divided into two different groups for pharmacokinetic investigations. Each of them received a single intravenous injection of 10 mg/kg DOG/MOFA/ICG nanoparticles or 2.84 mg/kg free GEM (the free GEM administered concentration was consistent with that contained in the DOG/MOFA/ICG nanoparticles). At predetermined time intervals , the blood samples were collected, which were precipitated and purified with acetonitrile. The content of GEM or DOG in all samples was measured using a UFLC-MS/MS method. 1 μL sample was injected into an ultrafast liquid chromatography system , equipped with Acquity UPLC BEH C18 column . The concentrations were analyzed by AB SCIEX Qtrap 5500 mass spectrometer, with electrospray positive mode. The reported concentrations were mean ± SD values from 5 mice/time point/group, and DAS 3.0 software was used to fit pharmacokinetic parameters.All p < 0.05 was considered to indicate a statistically significant difference.Statistical analyses were performed using GraphPad Prism 8.0 . All data were analyzed by Student’s t-test and one-way analysis of variance (ANOVA). All data are expressed as mean ± standard deviation and obtained from at least three independent experiments. 1H NMR , δ 7.77 , 7.51 , 6.45 , 5.46–5.21 , 4.58–4.12 , 2.56–2.30 , 2.16–1.90 , 1.76–1.53 , 1.32–1.24 , 0.88–0.86  .  . HRMS  δ 8.95 , 7.81 , 6.21 , 5.68–5.49 , 5.45–5.22 , 4.57–4.30 , 2.84–2.76 , 2.39–2.22 , 2.08–1.95 , 1.74–1.57 , 1.39–1.20 , 0.94–0.83  .  . HRMS  δ 8.92 , 7.76 , 6.18 , 5.72–5.45 , 4.90–4.68 , 2.52–2.26 , 1.76–1.56 , 1.42–1.18 , 0.95–0.83  . HRMS (ESI/TOF-Q): calcd. for [C37H63F2N3O6+H+] 684.4758; found 684.4760 .White solid (yield 68.3%). m, 5 H) . HRMS  δ 8.92 , 7.76 , 6.20 , 5.40–5.07 , 4.56–4.25 , 2.45–2.24 , 1.76–1.54 , 1.33–1.15 , 0.94–0.84  .  . HRMS  δ 8.93 , 7.02 , 6.18 , 5.44–5.23 , 4.70–4.54 , 2.48–2.28 , 1.71–1.62 , 1.35–1.25 , 0.95–0.82  . HRMS (ESI/TOF-Q): calcd. for [C23H35F2N3O6+H+] 488.2567; found 488.2571 .White solid (yield 48.8%). m, 6 H) . HRMS  δ 10.25 , 9.14 , 8.87 , 7.67–7.52 , 6.89–6.80 , 6.63 , 6.28 , 4.55 , 4.39 , 4.15–4.13 , 2.43–2.29 , 2.25–2.09 , 1.68–1.51 , 1.48–1.37 , 1.32–1.23 , 0.94–0.80  .  . HRMS  were screened to prepare prodrug self-assembled nanoparticles by reprecipitation method . As showThe average particle size of DOG, DOG/MOFA, DOG/ICG, and DOG/MOFA/ICG nanoparticles measured by the laser particle size analyzer were 94.74 ± 1.39 nm, 95.23 ± 0.89 nm, 102.6 ± 0.55 nm, and 109.2 ± 3.05 nm, respectively. The PDI of DOG, DOG/MOFA, DOG/ICG, and DOG/MOFA/ICG nanoparticles were 0.245 ± 0.010, 0.217 ± 0.006, 0.215 ± 0.011 and 0.188 ± 0.017, respectively . The pot1H NMR spectrum  and ν(C=O) (∼1750 cm−1) signals of DOG, ν(N-H)  signals of MOFA as well as ν(S=O)  of ICG, a low-wavenumber shift of ν(C=N) , two high-wavenumber shifts of ν(S=O)  and ν(N-H)  with the weaken signal of ν(C=O) (∼1750 cm−1) could be observed in DOG/MOFA/ICG nanoparticles . As depicted in It was found that the zeta potential and size of different nanoparticles in PBS (pH 7.4), 10% FBS solution, and DMEM under 4°C and 37°C almost remained unchanged within 7 days respectively . MoreoveAs shown in 2) for 5 min, the temperature of water, DOG, and DOG/MOFA nanoparticles hardly increased. The temperature of free ICG reached about 50°C, while the temperatures of DOG/ICG and DOG/MOFA/ICG nanoparticles reached 51.9°C and 57.0°C, respectively  was observed, which shows that these different nanoparticles had good biocompatibility for profiles .ICG-encapsulated DOG/ICG and DOG/MOFA/ICG nanoparticles enable cell imaging due to the autofluorescence of ICG, which was used to qualitatively analyze the cellular uptake of DOG/ICG and DOG/MOFA/ICG nanoparticles by fluorescence microscope. The free ICG was used as a control. As displayed in In vitro cytotoxicity of self-assembled nanoparticles against A549 cells was assessed by CCK-8 assay. The cell viability of A549 cells decreased with increasing drug concentrations in different self-assembled nanoparticles, and laser irradiation caused a further decrease in their viability . The results demonstrated DOG/MOFA/ICG nanoparticles possessed excellent antitumor activity via synergetic chemo-photothermal therapy.As illustrated in 1/2) of DOG in plasma was 5.66 ± 0.81 h, which was significantly longer than that of the free GEM group (3.08 ± 0.68 h). Furthermore, at 24 h after DOG/MOFA/ICG nanoparticles treatment, it was found that the plasma concentrations of DOG were higher than that of GEM, which indicated that DOG/MOFA/ICG nanoparticles administrated intravenously could have a sustained-release effect to maintain a long-term anti-tumor activity.To explore whether DOG/MOFA/ICG nanoparticles could ameliorate the pharmacokinetics properties of free GEM, the pharmacokinetic studies of free GEM and DOG/MOFA/ICG nanoparticles were conducted. The mice were given the free GEM or DOG/MOFA/ICG nanoparticles intravenously at an equivalent dose of 2.84 mg/kg GEM. At multiple time points, the plasma concentrations of free GEM and DOG were measured. As found in 1H NMR spectroscopy and TOF-Q mass spectroscopy. From the integral ratio of the methine protons (δ 7.79 and 7.53 ppm) in the pyrimidine ring and methyl protons (δ 0.90 ppm) in fatty acid, it could be proved that two oleic acid chains were coupled to the GEM successfully. Comparing the 1H NMR spectrum of free GEM and amphiphilic prodrugs, it was found that the signs of 3′-OH (δ 6.24 ppm) and 5′-OH (δ 5.20 ppm) disappeared, the chemical shifts of 3′-H and 5′-H of sugar moiety shifted to 5.39 and 4.43 ppm from 4.14 ppm as well as 3.73 and 3.63 ppm respectively, which verified that two oleic acid chains were coupled to 3′-OH and 5′-OH of sugar moiety  for 5 min, the temperature of free ICG reached about 50°C, while the temperatures of DOG/ICG and DOG/MOFA/ICG nanoparticles reached 51.9°C and 57.0°C respectively, which suggested the photothermal efficiency of nanoparticles loading ICG was significantly improved  was observed in the nanoparticles compared to the proportion of hemolysis in GEM (approximately 35%) , and theM itself . These rM itself . Thus DOin vivo as well as preferential cell uptake and cytotoxicity due to the active targeting of folate. This co-self-assembly of therapeutic, targeting, and photothermal agents is able to be used for the design of highly simplified and efficient integrated nanosystems for active targeting cancer synergistic therapy.A new folate-receptor-targeted carrier-free GEM nanoparticle loading indocyanine green for chemo-photothermal combination therapy was developed by the co-self-assembly between chemotherapeutic prodrug, active targeting agent, and photothermal agent. The readily available and highly reproducible DOG/MOFA/ICG nanoparticles showed monodispersity, high drug payload, excellent stability in physiological solutions, high photothermal conversion efficacy, the pH-/photo-responsive release of the drug in solution, sustained release activity, and higher bioavailability compared with free GEM


Cordia (Boraginaceae family) are widely distributed in the tropical regions of America, Africa, and Asia. They are extensively used in folk medicine due to their rich medicinal properties. This review presents a comprehensive analysis of the isolation, structure, biogenesis, and biological properties of quinones from Cordia species reported from 1972 to 2023. Meroterpenoids were identified as the major quinones in most Cordia species and are reported as a chemotaxonomic markers of the Cordia. In addition to this property, quinones are reported to display a wider and broader spectrum of activities, are efficient scaffold in biological activity, compared to other classes of compounds reported in Cordia, hence our focus on the study of quinones reported from Cordia species. About 70 types of quinones have been isolated, while others have been identified by phytochemical screening or gas chromatography. Although the biosynthesis of quinones from Cordia species is not yet fully understood, previous reports suggest that they may be derived from geranyl pyrophosphate and an aromatic precursor unit, followed by oxidative cyclization of the allylic methyl group. Studies have demonstrated that quinones from this genus exhibit antifungal, larvicidal, antileishmanial, anti-inflammatory, antibiofilm, antimycobacterial, antioxidant, antimalarial, neuroinhibitory, and hemolytic activities. In addition, they have been shown to exhibit remarkable cytotoxic effects against several cancer cell lines which is likely related to their ability to inhibit electron transport as well as oxidative phosphorylation, and generate reactive oxygen species (ROS). Their biological activities indicate potential utility in the development of new drugs, especially as active components in drug-carrier systems, against a broad spectrum of pathogens and ailments.Plants of the genus  Cordia .Sch.Sch75) wC. alliodora. According to them, the isolation of cordiachromene A (57) from C. alliodora confirms the presence of geranylphenol (76) as a precursor of compounds isolated from C. alliodora , cordiaquinol C (36), globiferin (45), alliodorin (46), and elaeagin (66), isolated from C. globifera, against KB (human epidermoid carcinoma of the mouth), BC-1 (human breast cancer cells), NCI-H187 , and Vero cell lines (African green monkey kidney fibroblast cells), were carried out. Compounds 2, 3 and 36 exhibited activity against the cell lines mentioned above with IC50 values ranging from 0.2 μM to 6.9 μM, while globiferin (45) was active only against NCI-H187 cells with an IC50 value of 0.5 ± 0.04 μM  and tumoral  cell lines. It showed high cytotoxic activity on human leukemic cancer cells and normal leukocytes with IC50 values of 11.2 and 6.8 μM, respectively while exhibiting IC50 values above 16.5 μM against the remaining cell lines  and Meglumine antimoniate , as reference drugs respectively -benzene-1,4-diol (43), and 6--2-methoxy-7-methylnaphthalene-1,4-dione (44) induced inflammation against RAW 264.7 macrophage cells by reducing cells viability with IC50 range value 71.66 ± 15.44–609.48 ± 5.05 μM. Lipopolysaccharide production was evaluated by inducing oxide nitric in RAW 264.7 cells. Among these compounds, 10,11-dihydrofuran-1,4-dihydroxyglobiferin (42) exhibited the best inhibition of NO (Nitric Oxide) synthesis with IC50 50.34 ± 9.88 μM, followed by compounds 44 (66.73 ± 10.28 μM) and 43 (105.83 ± 5.09 μM); the rest produced weak inhibition to induced inflammation against RAW 264.7 macrophage compared to dexamethasone (IC50 1.79 ± 0.04 μM) used as a positive control [Five meroterpenoids  was very active in inhibiting paw edema induced by a carrageenan injection, with a 57% and 60% reduction three hours after a dose of 10 and 30 mg/kg body weight, respectively [Ferreira et al. examined the anti-inflammatory activity of the water-soluble fraction of the heartwood methanolic extract of ectively .C. oncocalyx revealed that oncocalyxone A (18) could inhibit the growth of Gram-positive and Gram-negative pathogenic strains, even clinical specimens. It was more sensitive to Staphylococcus species than to Enterococcus, Listeria, Acinetobacter, and Stenotrophomonas species with an MCI range from 9.43 μg/mL to 151 μg/mL, and it showed high sensitivity against S. epidermidis (ATCC 12228™) with MIC 9.43 μM compared to vancomycin (MCI 1 μM) used as reference[S. aureus MED 55 (MIC 18.87 μM), S. aureus COL and S. epidermidis 70D (MIC 37.75 μM); and E. faecalis ATCC512999™ (MIC 75.5 μM) [Previous biological evaluation of reference. It also75.5 μM) S. aureus MED 55 strain  [It showed inhibition of biofilm production by ⁓70% in methicillin-resistant pecimen) C. rothii roots. The n-hexane fraction, which contained cordiachrome C (3), exhibited weak antibacterial activity against Gram-positive and Gram-negative bacteria. While the MeOH marc extract containing cordiaquinol C (36) and cordiachromene A (57) showed good antibacterial activity against Staphylococcus epidermidis with a minimum inhibitory concentration (MIC) 250 μg/disk, EtOAc marc extract containing cordiol A (55) was inactive against all the tested bacteria [Khan et al. examined the antimicrobial and antioxidant activities of the GC–MS profile fractions of bacteria .C. rothii roots have good activity with EC50 93.75 μM than n-hexane extract, which showed weak activity with EC50 187.5 μM [Regarding the antioxidant activity of these extracts, MeOH and EtOAc marc left extract of 187.5 μM .C. oncocalyx. The quinone fraction ) was evaluated in a rat model with CCl4-induced hepatotoxicity and the prolongation of pentobarbital sleeping time in mice by measuring plasma GPT and GOT. Only the quinone fraction inhibited the GPT level significantly (29%) with a 30 mg/kg dose. It also caused a significant reduction (45%) of CCl4-induced prolongation of pentobarbital sleeping time with a dose of 10 mg/kg. It confirmed the hepatoprotective effect involving free radical and lipoperoxidation and correlated with the antioxidant properties of quinones [Previous biological studies examined the antioxidant activity of the methanol extract of the heartwood of quinones . The latquinones . Moreovequinones ; this isquinones .3) and globiferin (45) showed significant antimycobacterial activity with MIC 1.5 and 6.2 μg/mL, respectively, while cordiachrome B (2) (12.5 μg/mL), cordiaquinol C (36) (25.0 μg/mL), diacetylcordiaquinol C (82) (25.0 μg/mL), alliodorin (46) (12.5 μg/mL), and elaeagin (66) (12.5 μg/mL) displayed weak activity compared to Rifampicin (0.0047 μg/mL), Isoniazid (0.05 μg/mL), and Kanamycin (2.5 μg/mL) used as standard drugs [In addition, cordiachrome C (rd drugs .3), cordiaquinol C (36), and diacetylcordiaquinol C (82) were evaluated for antimalarial activity against Plasmodium falciparum using dihydroartemisinin (IC50 0.0012 μg/mL), used as reference. They exhibited significant activity with IC50 0.2 ± 0.1 μg/mL, 0.3 ± 0.0 μg/mL, and 0.4 ± 0.1 μg/mL respectively, more than cordiachrome B (2) (IC50 1.5 ± 0.2 μg/mL), globiferin (45) (IC50 2.1 ± 0.5 μg/mL), alliodorin (46) (IC50 3.1 ± 0.5 μg/mL), and elaeagin (66) (3.6 ± 0.1 μg/mL) [Cordiachrome C (1 μg/mL) .Silva et al. evaluated the hemolytic activity of oncacalyxone A (18) through erythrocyte damage due to hemoglobin release. The compound did not show activity at the tested concentrations ≥ 151 μg/mL .21, 24, 30, 32, and 33 from C. polycephala roots were evaluated for hemolytic activity in mice erythrocytes. None was active with EC50 > 500 μmol L−1 [Compounds 9–18) isolated from C. oncocalyx by mice vas deferens bioassay. Compounds 10, 11 and 14 significantly inhibited the neurogenic contraction by 76%, 69%, and 63%, respectively, whereas compounds 12 and 15 did not considerably affect neurogenic contraction. Compounds 9, 10, 14, 16, 17 and 18 showed a completely reversible neuroinhibitory effect upon adding the pharmacological antagonist Promethazine and a partial reversible effect by yohimbine. Neurogenic contraction induced by compound 11 was irreversible by adding naloxone, famotidine, promethazine or yohimbine antagonists. However, compounds 9, 10, 14, 16, 17 and 18 did not inhibit neurogenic contractions using the ODQ, famotidine or naloxone antagonists. The authors found that reversible action may be related to pre-synaptic terminal and pre-synaptic receptor inhibition due to the co-release of histamine and norepinephrine [Matos et al. (2017) examined the neuroinhibitory effect of different compounds . To improve the number of quinones isolated from Cordia species, pressurized liquid extraction (PLE) could be used. [Although previous reviews reported different isolation methods and biological activities of be used. . Pressurbe used.  and dry be used. , 112.Cordia quinones from 1972 to 2023. The research shows that over 70 (1–70) quinones have been isolated from different parts of Cordia species with different skeletal structures. Meroterpenoid quinones were the major class of compounds isolated, with meroterpenoid benzoquinones being the most predominant in most species. The biosynthesis of Cordia quinones is not yet well understood, but the biogenesis and some biosynthetic pathways have been proposed to explain the presence of quinones in the Cordia genus.Using Cordia species in traditional medicine to treat various diseases has increased interest in their phytochemistry. This review presents the collective phytopharmacological information on Cordia species. This review discussed the potential of the genus Cordia as a promising source of new bioactive compounds that can provide quinones for various pharmaceutical applications.The extracts and isolated quinones demonstrated antimalarial, antimicrobial, anti-inflammatory, antibiofilm, antioxidant, antimycobacterial, antileishmanial, larvicidal, hemolytic, neuroinhibitory, and cytotoxicity properties. Most studies reported cytotoxicity against particularly cancer cell lines. It may be due to the ethnomedicinal uses of these species and the anticancer properties of the quinones. Although the biological activities of compounds can often be related to their structures, there is currently little information available to explain structure–activity relationships for the quinones occurring in