Then, the antitumor activity and systemic drug distribution of the implants were tested in a K7M2 OS bearing mouse model. The scanning electron microscope images showed that DOX was uniformly dispersed in the polymer matrix. Both the in vitro and in vivo release profiles of implants are characterized by three-phase release. Implantation of DOX-loaded implants into tumors can inhibit tumor growth in a dose-dependent manner. The pharmacokinetic behavior shows that intratumor chemotherapy through implants has a much higher drug concentration in tumors than in normal tissues, which may be the reason for improving antitumor activity and reducing systemic side effects. In summary, the drug release of the implants prepared in this study is sustained and stable, which promotes long-term local accumulation of drugs in tumors, improves the efficacy of chemotherapy and has low toxicity to normal tissues.The traditional systemic chemotherapy through intravenous infusion of doxorubicin (DOX) has many side effects. The aim of this study was to develop a PLGA-based DOX-loaded implant and to evaluate the efficacy and drug metabolism distribution of the implant in intratumoral chemotherapy for osteosarcoma (OS). In this study, implants containing DOX, poly( GLIADEL® has demonstrated excellent efficacy in the treatment of recurrent malignant gliomas  is an anthracycline discovered in the 1960s with anti-tumor effects. It is also one of the most common and effective chemotherapy drugs for OS. Direct insertion of drugs into DNA to affect chromatin, damage topoisomerase-II-mediated DNA repair, and generate free radicals that cause cell damage are the main mechanisms of DOX , and polyethylene glycol 4000 (PEG4000) by the melt-molding method. The implants were characterized in terms of drug content uniformity, micromorphology, drug–excipient compatibility, drug release, and degradability. Then, the antitumor activity of the implants was tested in a K7M2 OS-bearing mouse model. To investigate the pharmacokinetic characteristics of DOX-loaded implants after intratumoral implantation, an accurate and reliable ultra high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was used to detect drug concentrations in tumors, plasma and organs. Finally, the safety of the implant for intratumoral chemotherapy was verified by detecting blood biochemical indicators. The results of this study suggest that continuous intratumoral chemotherapy with DOX-loaded implants can effectively inhibit tumor growth and that increasing the dose of the drug results in higher tumor suppression rates (TSRs) without additional systemic toxicity.To overcome the limitations of DOX in clinical application, we constructed a biodegradable implant containing DOX, poly. Glibenclamide was purchased from Sigma-Aldrich . Formic acid was purchased from Tianjin Kermel Chemical Reagent Co., Ltd. . Acetonitrile and methanol were purchased from Merck Millipore . All of the solvents and reagents for analysis were HPLC grade.Doxorubicin hydrochloride injection was purchased from Hanhui Pharmaceutical Co., Ltd. . Doxorubicin hydrochloride  was obtained from Zhejiang Hisun Pharmaceutical Co., Ltd. . PLGA  is produced by Hefei Zhongren Science and Technology Co., Ltd. . Polyethylene glycol 4000 was purchased from Nanjing Well Pharmaceutical Co., Ltd. . For the cell culture, Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries2.2.Specific pathogen-free grade Kunming mice , Sprague-Dawley rats , and BALB/c mice  were purchased from Experimental Animal Center of Anhui Medical University . All animals are kept in a constant temperature (25 ± 2 °C) and 70 ± 5% relative humidity environment. All animal experiments were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Anhui Medical University .2.3.The implants containing DOX, PLGA, and PEG4000 (8:15:2 w/w/w) were prepared by the melt-molding method under aseptic conditions  and the actual drug content of each implant was calculated.2.4.2.The surface and cross-sectional morphology of DOX-loaded implants, blank implants, and implants after intratumoral chemotherapy  were observed using a Zeiss GeminiSEM300  scanning electron microscope. The images were obtained at 2.5 kV or 5.0 kV accelerating voltage. The samples were coated with gold using a CRESSINGTON 108 automatic sputter coater at 20 mA for 40 seconds before imaging.2.4.3.in vitro release characteristics of the implants were detected by the rotating basket method. Fifty milligrams of DOX-loaded implants were placed in a 200 mL release medium containing 0.01 mol/L Tris–HCl buffer (pH = 4.0), and the temperature of the release medium was constant at 37 °C ± 0.5 °C. At predetermined time points , the DOX content in the 5 mL release medium removed from the dissolution apparatus was determined by HPLC. Then, 5 mL of fresh release medium was added back to the dissolution flask.The 2.4.4.in vivo release characteristics of the DOX-loaded implants. The removed implant was rinsed with deionized water and dried, and then the drug content remaining in the implant was measured by HPLC. The in vivo cumulative release percentage of DOX was calculated as follows:The implants implanted in the muscles of Kunming mice were taken out at predetermined time points  to test the 2.4.5.−1 and use high-purity nitrogen (100 mL/min) as the purge gas.The DSC analysis of the DOX-loaded implant was performed by a thermal analysis instrument Q2000 . First, samples (4–5 mg) of DOX-loaded implants, PLGA, PEG4000, and DOX were sealed in aluminum pans. The parameters of DSC analysis are set to detect in the temperature range of 20–260 °C at a heating rate of 10 °C min2.4.6.−1. Each generated spectrum was the result of 32 scans with a resolution of 4 cm−1.Potassium bromide was added to the DOX-loaded implants, PLGA, PEG4000, and DOX samples, and then they were made into slices by the tableting method. The infrared spectrum of the slices was generated by an FTIR spectrophotometer  with a coverage range of 400–4000 cm2.4.7.At predetermined time points , the blank implants (without DOX) implanted into the muscles of Sprague-Dawley rats were taken out. The removed implant was weighed after being vacuum dried. The percentage of weight loss was calculated according to the formula:2.4.8.The HPLC system  was equipped with two LC-15C pumps, a SPD-15C essential UV detector, and a CTO-15C essential column oven. A Hypersil BDS C6H5 column  was used as the analytical column. The mobile phase consisted of sodium dodecyl sulfate (SDS) solution (1.44 g SDS and 0.68 mL phosphoric acid co-dissolved in 500 mL ultra-pure water), acetonitrile and methanol . The HPLC detection parameters were as follows: the column oven temperature was set at 25 °C; the injection volume was 10 μL; and the ultraviolet (UV) spectrum wavelength was 254 nm. The external standard method was used for quantitative analysis.2.5.2.5.1.6 K7M2 cells were resuspended in 100 μL PBS and injected subcutaneously into the right forelimb axilla of each BALB/c mouse . A palpable tumor appears within 14 days and the in vivo study starts when the tumor volume reaches 250–300 mm3.The K7M2 OS cell line was purchased from Wuhan Procell Life Technology Co., Ltd. . The K7M2 OS cells were cultured in DMEM medium containing 10%FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Before the experiment, procedures such as hair shaving and skin disinfection were performed on the tumor site. 5 × 102.5.2.n = 6 per group): blank implant group, DOX solution group , DOX implants-L group , DOX implants-M group , DOX implants-H group , and DOX implants-Ultrah group . Calculate the dosage of DOX solution in mice according to the usage of DOX (80 mg/m2) in clinical chemotherapy for patients with OS ). During the treatment, the tumor volume was measured every two days using a digital vernier caliper. Tumor volume (V) was calculated by the formula V = 0.5×L×W2, in which L and W meant the length and width of the tumor, respectively .2.7.At the end of the experiment, the mice were sacrificed to collect organs  and tumor tissues. The tissue was immersed in 10% neutral paraformaldehyde for 24 hours. The dehydrated tissue was embedded in paraffin and cut into 4 μm thick tissue sheets. Tissue sections were stained with hematoxylin and eosin for histopathological examination.2.8.We established a UPLC–MS/MS method to quantitate DOX in plasma and tissues of K7M2 OS-bearing mice. The performance of the UPLC–MS/MS method was fully validated following previous reports, including selectivity, calibration curve, accuracy, precision, recovery, matrix effect, carry-over, dilution integrity, and stability  and DOX implants group . At predetermined time points , the plasma, heart, liver, spleen, lung, kidney, and tumor of the mice were separated and collected to detect the drug concentration in the blood and tissue. Five mice were set at each time point.Forty-five K7M2 OS bearing mice were randomly divided into two groups: DOX solution group  working solution  was added to 100 μL of plasma and vortex mixed for 30 seconds. Then, 400 μL 0.1% (v/v) formic acid-acetonitrile was added to the above solution and vortex mixed for five minutes. After the mixed liquid was centrifuged at 13,000 rpm for 10 minutes, 5 μL of the supernatant was taken for further analysis.Mouse tissues  weighing 30 mg were homogenized in 1 mL ultrapure water containing 0.1% (v/v) formic acid for 15 minutes. After the homogenate was centrifuged at 5000 rpm for 10 minutes, 20 μL IS working solution  was added to 100 μL of tissue homogenate and vortex mixed for 30 seconds. The subsequent processing procedure was the same as the plasma samples.2.8.3.2.8.3.1.The LC–MS/MS analysis was performed using an AB Sciex 5500 mass spectrometer coupled with an ExionLC high-performance liquid chromatography system .2.8.3.2.Column type: Acquity UPLC BEH C18 . Mobile phase: the mobile phase was composed of ultrapure water (phase A) and acetonitrile (phase B), both adding 0.1% of formic acid (v/v). Gradient elution program: 0 min, 5% B; 0.5 min, 5% B; 1.2 min, 95% B; 2.2 min, 95% B; 3.0 min, 5% B; 4.0 min, 5% B. Flow rate: 0.2 mL/min. The column oven and autosampler temperature were set to 37 °C and 10 °C, respectively.2.8.3.3.m/z 544.2 → 397.2 and 494.2 → 369.2).The optimized parameters for the mass spectrometric analysis were the following: capillary voltage, 2.7 kV; cone voltage, 20 V; desolvation gas flow rate, 650 L/hours; desolvation gas temperature, 350 °C; collision energies, 15 eV. The mass detection was used in the positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) for the quantification of DOX and its IS , and the data were expressed as mean ± standard deviation (SD). The comparison between the multiple groups uses one-way ANOVA, followed by Dunnett’s test to detect intergroup differences. *3.3.1.Figure S1(A)). The average weight of the implants was (3.31 ± 0.19) mg (n = 10). Five implants were selected to detect their actual drug content by HPLC. The results showed that the average actual drug content of the implant was (29.78%±0.66%), which was close to the number in the label claim of the drug  (Table S1) (The DOX-loaded implants prepared by the melt-molding method were further molded into a cylinder with an average diameter of (0.86 ± 0.03) mm and a length of (4.22 ± 0.26) mm (able S1) .3.2.SEM was used to observe the microstructure and degradation characteristics of the DOX-loaded implants. SEM found that the surface of the DOX-loaded implant was homogeneous without obvious holes and channels. The comparison of the blank implant and the DOX-loaded implant showed that the drug was evenly distributed in the implant . As the 3.3.in vitro and in vivo release curves of the DOX-loaded implant were three-phase release, which was characterized by the initial burst release followed by continuous release of DOX, and then enters the plateau phase. The in vitro release results showed that the implant released about 26.4% of DOX within two hours .Compared with the control group, the myocardial enzyme detection results showed that only the DOX-loaded implants (156 mg/kg) caused the increase of LDH, CK, and CK-MB in mice . FurtherFigure S2).To further evaluate the systemic toxicity of DOX-loaded implants on the mice, histopathological studies were performed on the major organs of K7M2 OS bearing mice. Based on histopathological observation , a sligh3.9.Figure S4). No peaks were observed at the elution area of the target analyte in the blank plasma and tissue samples .The DOX concentration was measured by UPLC–MS/MS using the IS method. By measuring the standards of DOX and IS, the retention times of DOX and IS were determined to be 2.58 minutes and 3.90 minutes, respectively . Moreover, the histopathological examination supported the biosafety of the DOX-loaded implant in major organs . All results demonstrated that the DOX-loaded implants were free of systemic toxicities, offering great promise for clinical applications in OS therapy.All mice died within five days after receiving the intraperitoneal injection of DOX solution (19.5 mg/kg). In this study, DOX was rapidly absorbed into the systemic circulation after intraperitoneal injection, which resulted in the drug being widely distributed in the organs in extremely high concentrations within six hours. The reason for the death of mice may be that the extremely high drug concentration in a short period caused organ damage  the implant has excellent biodegradability, avoiding the second operation to remove the residual excipient. (ii) The drug and excipients have excellent compatibility. (iii) The implant directly releases the drug inside the tumor can enhance the anti-tumor effect of DOX. (iv) The DOX-loaded implants can improve the biodistribution of the drug and reduce systemic side effects by limiting the accumulation of the drug in normal tissues. (v) The implant can keep the drug at an effective therapeutic concentration for a long time in the tumor.5.In conclusion, we report a biodegradable implant for localized and sustained delivery of DOX to treat OS. The DOX-loaded implants we prepared in this study provided a high local DOX concentration, sustained and stable drug release, extended drug retention inside of tumor, and low toxicity to normal tissues, especially cardiac toxicity. The DOX-loaded implants could significantly inhibit K7M2 OS growth in a dose-dependent manner. Therefore, we conclude that the DOX-loaded implants are a promising drug delivery system in terms of preoperative adjuvant chemotherapy and the prevention of tumor recurrence.Click here for additional data file.


Asarum (Xixin), which contains analogues of aristolochic acid (AA), is the only species of the genus Aristolochia included in the Chinese Pharmacopoeia 2020. However, the contents and nephrotoxic effects of AA analogs in Asarum (Xixin) and its formulations have not been clarified. An automatic, effective solid phase extraction process and UPLC-MS/MS method were established for the pretreatment and quantitative detection of AA analogues in commercially available traditional Chinese patent medicines. The cytotoxicity and DNA damage induced by five analogues of AA were evaluated by CCK8 using human kidney cells (HK-2) and comet assays. HPLC was used to detect the analogues of AA in Asarum heterotropoides F. Schmidt (Xixin). The results showed that the contents of AA I, AA II, and AA IIIa were below the detection limit, while AA IVa and AL I presented relatively high contents of Asarum heterotropoides F. Schmidt (Xixin), within the range of 66.50–121.03 μg/g and 19.73–43.75 μg/g, respectively. The levels of AA analogues were in the nanogram-per-gram level in the main traditional Chinese patent medicines. AA I and AL I exhibited relatively high cytotoxicity at 48 h in CCK8 assays, while AA II, AA IIIa, and AA IVa showed weak cytotoxicity even at 800–1,000 μM. AA I induced significant pathological alterations and direct DNA damage at 40 mg/kg and 20 mg/kg, respectively. No distinct nephrotoxicity or hepatotoxicity was observed in mice treated with AA II, AA IIIa, AA IVa, or AL I at 40 mg/kg in this study. Consumption of Asarum heterotropoides F. Schmidt (Xixin) with controlled doses and periods is relatively safe as the contents of AA analogues in Asarum heterotropoides F. Schmidt (Xixin) and its formulations were far below those causing acute toxicity in this study. But, the long-term toxicity of Asarum heterotropoides F. Schmidt (Xixin) still needs further study. Exposure to AA is associated with nephropathy and upper tract urothelial carcinoma (UUC) (Aristolochia clematitis (Aristolochia species (in vitro and in vivo (Aristolochic acid (AA) exists mainly in plants of the Aristolochiaceae family that have been used in pharmaceutical formulations for hundreds of years to treat various diseases . Exposurma (UUC) . AA is aematitis . There aematitis . Among t species . AA I an species . The add in vivo .Aristolochia plants have been prohibited for clinical use in many countries and districts except Asarum (Xixin), as it contains relatively low levels of AA I and AA II (Asarum (Xixin) products are not cytotoxic in HK-2 cells in vitro  has aroused public concern.In 2002, AA I was listed as class I carcinogen by WHO due to its nephrotoxicity and carcinogenicity. Subsequently, most nd AA II . Furtherin vitro . Recentlin vitro . The forr models . AA I car models . AlthougAsarum (Xixin) on the Chinese mainland. Since AA I can induce an exceptionally long-term persistence of DNA adducts and irreversible nephrotoxicity, the content of AA I should be strictly controlled (Asarum (Xixin) from natural sources and its formulated products are not clear. Besides the trace amounts of AA I and AA II, there are many other AA analogues in Asarum (Xixin)  . Whether (Xixin) .Asarum heterotropoides F. Schmidt (Xixin) and its formulated products. The other was to assess their in vitro and in vivo toxicities, to provide a rationale for the clinical application of Asarum heterotropoides F. Schmidt (Xixin). HPLC was usually used for the detection and quantification of AA analogs in crude drugs (Asarum heterotropoides F. Schmidt (Xixin) of different origins and commercially available traditional Chinese patent medicines. Cytotoxicity, acute toxicity, and comet assays were performed to compare the in vitro and in vivo toxicity of different AA analogues, to provide a theoretical basis for the safe clinical application of Asarum heterotropoides F. Schmidt and its formulated products.One aim of this study was to quantitatively detect the contents of AA I, AA II, AA IIIa, AA IVa, and AL I in de drugs . Howeverde drugs . In thisAsarum heterotropoides F. Schmidt (Xixin) samples were purchased from different districts of the Chinese mainland with batch numbers XX01–XX15. All were authenticated as the roots and rhizomes of Asarum heterotropoides F. Schmidt (Xixin) by Prof. Jinda Hao, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Traditional Chinese patent medicines containing herbs from the Aristolochiaceae family were collected from online and offline pharmacies.Fifteen batches of The AA I reference standard (purity >98%) was purchased from the National Institutes of Food and Drug Control . AA II, AA IIIa, AA IVa, and AL I (purities >98%) reference standards were obtained from Chengdu Push Bio-technology Co., Ltd. . Methanol, acetonitrile, formic acid, and ammonium acetate (HPLC and MS/MS grade) were purchased from Fisher Scientific . Aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase activities (ALP), creatinine (CREA), blood urea nitrogen (BUN), total bile acid (TBA), and total bilirubin (TBIL) were purchased from Hua Sin Science Co., Ltd .Asarum heterotropoides F. Schmidt (Xixin). The mobile phases were acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B). Separations were performed as follows: 0–5 min, 30–30% A; 5–5.01 min, 30–36% A; 5.01–26 min, 36–43% A; 26–28 min, 43–43% A; 28–28.01 min, 43–30% A; 28.01–30 min, 30–30% A. The flow rate was 1.0 ml/min. AA analogues were detected at a wavelength of 254 nm. The column temperature was maintained at 20–23°C. Samples (10 μl) were injected into the HPLC system.The Agilent Eclipse XDB C18 column  was used for chromatographic separations of the analogues of AA in Quantitative determination of analogues of AA in traditional Chinese patent medicines was performed with a UPLC-MS/MS system consisting of an ACQUITY UPLC I-Class coupled to a Xevo-TQS detector . Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column  with a flow rate of 0.3 ml/min at 35°C. Separation was obtained with the following gradient: 0–1.0 min, 10% B; 1.0–7.0 min, 10–75% B; 7.0–7.2 min, 75–95% B; 7.2–10.2 min, 95% B; 10.2–10.3 min, 95–10% B, 10.3–12.0 min, 10% B . Both mobile phases were added with 5 mM ammonium acetate and treated under ultrasonication for 5 min before usage. The injection volume was 1.0 μl.Multiple reaction monitoring mode (MRM) and positive electrospray ionization were applied for the quantitative determination. The instrument parameters were as follows: source temperature, 150°C; capillary voltage, 3.5 kV; desolvation gas flow, 800 L/h; cone gas flow, 150 L/h; nebulizer gas, 7.0 bar; desolvation temperature, 550°C. Details of the parent/daughter ions, cone voltages, and collision energies for AA I, AA II, AA IIIa, AA IVa, and AL I are listed in Asarum heterotropoides F. Schmidt (Xixin) samples were crushed into a powder and 1.0 g was placed in a conical flask, followed by cold immersion with 20 ml of 70% ethanol for 1 h and ultrasonic extraction  for 30 min. The supernatant was collected and the residue was extracted again according to the same procedures. Then the supernatants were combined and filtered through a 0.22-μm membrane prior to HPLC determination.g for 15 min before UPLC-MS/MS detection.Traditional Chinese patent medicines  were crushed and filtered through a 60-mesh sieve. Honeyed pill was cut into small pieces. Each 50.0 mg sample was accurately weighed and placed in a 10 ml centrifuge tube, to which 4 ml of methanol was added. The mixture was ultrasonicated for 90 min at room temperature and then centrifuged at 4,000 rpm for 15 min. The supernatant was collected and dried with nitrogen at 45°C. The residue was redissolved with 1.0 ml of 30% methanol and the pH was adjusted to 8.0–9.0 using aqueous ammonia. Biotage Extrahera  was applied for the automatic solid phase extraction process. Waters Oasis MAX Cartridge 3 cc/60 mg columns  were first activated by 3.0 ml of methanol and 3.0 ml of water. Then the columns were balanced with 3.0 ml of 30% methanol. The samples were loaded and washed sequentially with 5% aqueous ammonia and 60% methanol, followed by elution with 3.0 ml of methanol and 4.0 ml of methanol containing 8% FA. The eluent was collected and dried with nitrogen. Then the residue was redissolved with 1.0 ml methanol and centrifuged at 13,000×50 values were obtained by fitting the curve through nonlinear regression with GraphPad Prism 7.0 .Cytotoxicity analysis was determined using the Cell Counting kit 8 (CCK8) . HK-2 was cultured in DMEM/F12  at 37°C in 5% carbon dioxide incubator. The cells were maintained at 80% confluency and the medium was replaced every 3 days for routine cultivation. HK-2 cells  were seeded in 96-well plates overnight. The cells were then treated with analogues of AA for 24 or 48 h at different concentrations . Subsequently, the cells were incubated with 10% CCK8 solution for another 2 h. The final concentration of DMSO in the medium did not exceed 1.0% v/v. The absorbance (OD) of each well was measured with a spectrophotometer  at 450 nm and the ICg. AST, ALT, ALP, CREA, BUN, TBA, and TBIL levels in plasma were examined. SPSS 17.0  was used for the statistical and significance analyses. The renal and liver tissues were embedded and stained with H&E. Histological images were obtained through Olympus BX63  at a magnification of ×200.The animal experiments were approved by the Research Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences . The experiment was carried out according to ethical guidelines and regulations for the use of laboratory animals. A total of 160 ICR mice were randomly divided into 16 groups, each comprising 10 mice . The drugs were administered in a single dose by oral gavage and the dose volume was 0.2 ml/10 g. The control group was treated with 0.5% CMC-Na. Other groups received AA I, AA II, AA IIIa, AA IVa, or AL I, at concentrations of 10, 20, and 40 mg/kg, respectively. Animal weights were recorded every two days. The animals were fasted overnight before experiments and sacrificed on day 14. Blood samples were collected and centrifuged for 15 min at 3,500×n = 5 for each group) and administrated with saline, EMS (577.6 mg/kg), AA I (20 mg/kg), AA II (20 mg/kg), AA IIIa (20 mg/kg), AA IVa (20 mg/kg), or AL I (20 mg/kg) for three consecutive days, respectively. After anesthetization and perfusion, renal and liver tissues were obtained. Comet analysis was performed 2–6 h after the last lavage and the processes were according to Günter Speit . The method showed good separation and reproducibility. AA I, AA II, AA IIIa, IVa, and AL I were eluted at 24.36, 20.81, 9.36, 11.23 and 20.31 min, respectively. The quantitative results are shown in High-performance liquid chromatography (HPLC) was applied to determine the content of AA analogues  in rootsAristolochia debilis Siebold & Zucc. (Madouling) or Aristolochia mollissima Hance (Xungufeng), which have been reported to have a high content of AA I (Asarum heterotropoides F. Schmidt (Xixin), a species of the genus Aristolochia, the content of AA I was below 100 ng/g. AA IVa was identified in 86.6% of the samples and the concentrations were within the range of 0.005–1.287 μg/g. Variable content of AL I was detected in all traditional Chinese patent medicines, except for the Shenmei Yangwei and the Shenqi Jianwei Granules. The highest concentration of AL I was found in the Shaqi pill at 5.565 μg/g.An effective and automatic SPE method was established and successfully applied in the pretreatment of traditional Chinese patent medicines. Recovery of each analyte was within the range of 86.50–111.88% at three concentration levels . The con of AA I . As for Asarum heterotropoides F. Schmidt (Xixin) may account for the content differences of the AA analogues.Different dosage forms of traditional Chinese patent medicines with the same composition contained different amounts of AA analogues. For example, the contents of AA I, AA II, AA IVa and AL I in the Shensanqishangyao tablet were 0.020, 0.016, 0.023, and 0.034 µg/g, respectively. However, AA I or AA II were not detected in the Shensanqishangyao capsule. And the contents of AA IVa and AL I were 0.010 and 0.055 µg/g, respectively. The origins of the herb and the processing method of Asarum heterotropoides F. Schmidt (Xixin)–containing medicines. We questioned whether AA IVa and AL I could play a role in the nephrotoxicity of Aristolochia. Thus, we conducted both in vitro and in vivo toxicity assays. The cytotoxicity of the AA analogues was evaluated using the CCK-8 assay. HK-2 cells were treated with AA analogues for 24 or 48 h. AA analogues showed variable degrees of cytotoxic effects in HK-2 cells, with AA I and AL I exhibiting the strongest cytotoxicity  treated with 40 mg/kg AA I were dead on days 4, 5, and 6 due to the acute kidney failure and the survival rate was 30%. 10% of mice died (n =1 of 10) on day 8 in the group treated with AA II (40 mg/kg). There was no death occurring in control, AA I , AA II , AA IIIa , AA IVa , AL I  treated groups.Survival of the control and treated groups were monitored for 14 days. As shown in After mice were sacrificed, kidney and liver tissue were obtained for histopathological analysis. Significant lesions were only found in the renal tissues of mice treated with 40 mg/kg AA I. As shown in p < 0.001) and the olive tail moment was 7.45 ± 1.94 (p < 0.05), respectively , which has been used for centuries as an analgesic and antitussive (Asarum (Xixin) contains relatively low levels of AA I and AA II in roots and rhizomes; therefore, it is considered as a safe drug and is widely used in traditional Chinese medicines (Aristolochia has once again aroused public concern (Asarum (Xixin) is required to be <0.001% according to the Chinese Pharmacopoeia 2020, the contents of AA analogues in herbs and their commercial products are still not known. In this study, the analogues of AA were quantitatively determined in 15 batches of Asarum heterotropoides F. Schmidt (Xixin) and 44 traditional Chinese patent medicines. AA I, AA II, and AA IIIa were not detected in Asarum heterotropoides F. Schmidt (Xixin) samples. Relative levels of AA IVa and AL I were found in all samples with species and regional differences, which was in consistent with previous studies (Asarum heterotropoides F. Schmidt (Xixin) with less AA analogues content was an important way to improve the clinical safety of Asarum heterotropoides F. Schmidt (Xixin) and its formulated products. In this study, XX04 from Liaoning had some advantages on the low content of AA analogues. However, more samples of different species and origins need to be detected, in order to minimize the potential nephrotoxicity and hepatotoxicity.Plants of Aristolochiaceae have been gradually prohibited in the clinical use due to the nephrotoxicity and carcinogenicity of its component AA, with the exception of itussive . Asarum edicines . In rece concern . Althoug studies . Selecting/g in most samples. Although processing can reduce the content of Aristolochia AAs to varying degrees (Aristolochia debilis Siebold & Zucc. were still much higher than those with Asarum (Xixin). Thus, special attention should be paid to the potential nephrotoxicity or hepatotoxicity. The AA analogues varied in different dosage forms of the same prescription. Species and origins of Asarum heterotropoides F. Schmidt (Xixin) with different contents of AA analogues may be one of the reasons. The processing method also affected the content of active components, suggesting that optimizing processing may be another approach for reducing the potential toxicity.Traditional Chinese patent medicines contain different types of medical constituents and the complex matrix brings difficulties in quantitative determination. The validation of a previous method is usually estimated based on the analysis of spiked samples of selected products in different forms and compositions, which makes the determination complicated . In addi degrees , the conAristolochia species (Asarum heterotropoides F. Schmidt (Xixin) and its formulated products. Previous studies focused on the toxicity of AA I and AA II, while little is currently known about the toxicity of AA IIIa, AA IVa, and AL I, especially in terms of in vivo toxicity and carcinogenicity. To clarify whether these AA analogues contribute to the process of renal damage, both in vitro and in vivo toxicity assays were performed. Aristolochic acids I, II, IIIa, IVa, and aristolactam I showed varying degrees of cytotoxic effects to HK-2 cells, with AA-I and AL-I exhibiting the strongest cytotoxicity after 48 h of exposure. The localization of functional groups in the structure played a decisive role in cytotoxicity . Most traditional Chinese patent medicines in the study contained AA I, AA IVa, and AL I and the contents were at the ng/g level. Among the AA analogues studied, AA I exerted the strongest nephrotoxicity, while AA II, AA IIIa, and AA IVa showed weak toxicity both in vitro and in vivo. AL I showed significant cytotoxicity to HK-2 cells, but no renal toxicity was observed in vivo at 40 mg/kg. The contents of analogues of AA in Asarum heterotropoides F. Schmidt (Xixin) and its formulated products were far below the concentrations that induce acute nephrotoxicity; therefore, consumption of Asarum heterotropoides F. Schmidt (Xixin) with controlled doses and periods is relatively safe. However, considering the relatively high contents of AA IVa and AL I in Asarum heterotropoides F. Schmidt (Xixin), as well as the high cytotoxicity of AL I, the long-term toxicity of AA IVa and AL I should be further studied. In addition to the above-mentioned findings, the present study provides a basis for a broader quantitative determination of AA analogues and has a guiding implications for the safe clinical application of Asarum heterotropoides F. Schmidt (Xixin) and its formulated products.In conclusion, our study revealed that AA IVa and AL I were presented in relatively high levels in


P > 0.05). In conclusion, smoking is harmful to the cardiac system of young people, especially when nicotine content ≥4 mg dosage.Around the whole world, smoking is considered harmful to human health, such as increasing the risk of cardiovascular disease  and lung cancer. The purpose of this study was to explore whether nicotine, the main component of tobacco, has adverse effects on heart rate variability (HRV) in adolescents, so as to remind adolescents not to smoke and not to take pleasure in abusing nicotine. In this study, 40 male and 40 female young healthy nonsmoking subjects were selected to analyze the changes of HRV after taking 4 mg nicotine orally. We found that nicotine reduced HRV in young healthy male and female subjects, and there was no gender difference in this effect ( Smoking is an important and well-established risk factor for cardiovascular disease. Nicotine is a colourless, poisonous alkaloid, derived from the tobacco plant. It is the substance in tobacco to which smoker can become addicted. Although nicotine is thought to be harmful to human's health, people still use nicotine replacement therapy (NRT), especially Nicabate CQ lozenge, for smoking cessation as it is effective in quitting smoking. The response of nicotine is fast and excitatory in nature. The intake of nicotine will produce similar effects as smoking that increases the sympathetic activity to cause rapid heartbeat, increased blood pressure, rapid shallow breathing, hyperglycemia, and increased level of bad cholesterol-LDL .Heart rate variability (HRV) is the variation of time interval from beat to beat as a result of cardiac autonomic modulation (CAM). The imbalance in CAM is thought to be the causation of heart disease, and HRV measurement is widely accepted to reflect the cardiac autonomic modulation. HRV measurement  is well-In order to determine whether nicotine has an effect on HRV, and whether this effect is gender dependent, 80 young healthy students  were recruited in this research.Treatment: Nicabate CQ lozenge (GlaxoSmithKline) contains nicotine at 4 mg dosage and administered orally.Placebo: mint lozenge and ingested orally.2) and 40 age, weight, and BMI matched females  aged between 18 and 22 yrs, weighted 42-65 kg, with body mass index (BMI) within 18-23.5 kg/ms2, were recruited . All these adolescents had never suffered from cardiovascular disease, no implanted cardiac pacemaker, no hypertension (systolic pressure > 140 mmHg or diastolic pressure > 90 mmHg), no hypotension (systolic pressure < 110 mmHg or diastolic pressure < 60 mmHg), no diabetes, no allergy to nicotine, no any medication during or a week before the experiment, and avoid the female if she was in the menstrual period during the experiment.80 young healthy nonsmoking Chinese Han volunteers from Henan College of Finance and Taxation with native place of Henan Province, 40 males  of each domain and the total power of the three domains was recorded. LF power mainly represented sympathetic nervous system (SNS) activity and HF power mainly represented parasympathetic nervous system (PNS) activity [In this study, HRV was measured by a short-term (4-minutes) frequency domain method. This method measured the underlying variance (power) , 9 in thDue to individual differences, the total power of RR interval variance was different for each subject. LF power and HF power are part of the total power, so they cannot be directly used for analysis. Therefore, the LF power and HF power were normalized into LF (n.u.) and HF (n.u.) for analysis. LF (n.u.) and HF (n.u.) were referred to as HF and LF hereinafter .OMRON T8 Automatic Blood Pressure Monitor: measuring HR, systolic, and diastolic blood pressure.PowerLab 4/25 (AD Instruments): detecting and processing the ECG signal and transferring the ECG signal to the computer for viewing and analysis. Subjects sat in a relaxed upright posture; the ECG was detected by AD instruments using 3 electrodes applied to the body of the subject .Chart 5 for Windows : processing ECG signal and frequency-domain analysis .The examiners and the volunteers were both blind of the order in which the drugs were given or eaten. Every subject was asked to take either nicotine or mint tablet randomly in the first time attendance of the experiment from 15:30 pm to 17:30 pm; then, the other kind of drug was taken 1 week latter at the same time of the day. On the day of the experiment, each subject was asked to not eating and drinking except pure water after 7:30 am  and no a2), subject with BMI out of range 18.5-23.5 kg/ms2 excluded from this study; the initial blood pressure was measured to exclude subject who had hypertension or hypotension; the initial HR was measured to ensure the subject was settled down.Before starting the measurement, the information sheet and the consent form were signed; blood pressure and HR were measured. The height and weight were measured to calculate the BMI (body mass (kg)/height (m)The ECG was measured at a 4-minute interval, and blood pressure was measured after each ECG measurement section to ensure there was no hypotension or hypertension occurred during the experimental process. Three times of ECG were recorded: before nicotine intake, 10 min after nicotine intake, and 20 min after nicotine intake.T-test was performed to compare several HRV measurements between the three trials  to during and after.To see if there were any gender differences in nicotine effect: two-tail unpaired X, % change of his or her HR at experimental trail After = (HRAfter–HRBefore)/HRBefore, where HRBefore is the value of HR recorded at experimental trail before, i.e., the baseline of HR. % change of HF and LF was calculated in the same way.Calculation of % change in HF (nu), LF (nu), and HR: for an example of subject P < 0.05.Data in the result part was presented as mean ± SEM . Significance was chosen at P > 0.05). In male: LF varied from 64.63 ± 2.27 at before to 65.34 ± 2.54 at during and to 64.28 ± 2.04 at after; there was no any significant difference between any two of the three experimental trails (P > 0.05). Therefore, SNS activity was not altered by mint ingestion (LF (nu) analysis, in female: LF varied from 64.26 ± 2.81 at before to 65.68 ± 2.75 at during and to 63.78 ± 2.67 at after; there was no significant difference between any two of the three experimental trails (ngestion .P > 0.05). In male: HF varied from 28.11 ± 2.23 at before to 27.89 ± 2.33 at during and to 28.01 ± 1.82 at after; there was no any significant difference between any two of the three experiment trails (P > 0.05). Therefore, PNS activity was not altered by mint ingestion (HF (nu) analysis, in female: HF varied from 27.87 ± 2.56 at before to 27.31 ± 2.13 at during and to 28.14 ± 2.35 at after; there was no significant difference between any two of the three experiment trails (ngestion .P > 0.05). In male: LF/HF varied from 2.68 ± 0.30 at before to 2.72 ± 0.31 at during and to 2.66 ± 0.28 at after; there was no any significant difference between any two of the three experimental trails (P > 0.05). Therefore, the CAM did not significantly changed upon the mint ingestion in both females and males.LF/HF, in female: LF/HF varied from 2.97 ± 0.46 at before to 3.09 ± 0.43 at during and to 2.92 ± 0.41 at after; there was no significant difference between any two of the three experimental trails  (P < 0.05). Therefore, SNS activity was enhanced by nicotine ingestion and was significantly increased at 20 min after nicotine intake.LF (nu) analysis, in female: LF increased from 65.36 ± 2.61 at before to 67.06 ± 2.75 at during and to 69.95 ± 2.67 at after; there was only a statistically significant difference at after compared with before ( < 0.05) . In maleP < 0.05) (P < 0.05). Therefore, PNS activity was suppressed by nicotine ingestion, and it was significantly reduced at 20 min after nicotine intake.HF (nu) analysis, in female: HF decreased from 27.19 ± 2.3 at before to 25.4 ± 2.13 at during and to 23.9 ± 2.35 at after; there was only a statistically significant difference at after compared with before ( < 0.05) . In maleP < 0.05). In male: LF/HF varied from 2.67 ± 0.27 at before to 3.10 ± 0.38 at during and to 3.44 ± 0.40 at after; there was only a statistically significant difference between experimental trails after and before (P < 0.05). Therefore, the CAM was shifted to a SNS predominant manner after nicotine ingestion at 20 min after nicotine intake in both females and males.LF/HF analysis, in female: LF/HF varied from 3.09 ± 0.47 at before to 3.23 ± 0.40 at during and to 3.86 ± 0.54 at after; there was only a statistically significant difference between experimental trails after and before (P < 0.05). In male: HR increased from 72.65 ± 2.02 at before to 74.48 ± 2.27 at during and to 75.42 ± 2.25 at after; there was only a statistically significant difference at after compared with before (P < 0.05). Therefore, nicotine ingestion had significant effect on elevating HR in both male and females, especially at 20 min after nicotine intake.HR analysis, in female: HR increased from 78.06 ± 2.69 at before to 82.02 ± 2.92 at during and to 80.51 ± 2.86 at after; there were statistically significant differences at both during and after compared with before (P > 0.05). In male: SDNN varied from 93.24 ± 6.50 at before to 86.18 ± 7.05 at during and to 86.01 ± 5.87 at after; there was no significant difference between any two of the three experiment trails (P > 0.05). Therefore, nicotine ingestion had no effect on SDNN within 20 min after nicotine intake.SDNN analysis, in female: SDNN varied from 71.21 ± 5.12 at before to 72.98 ± 6.08 at during and to 72.58 ± 5.77 at after; there was no significant difference between any two of the three experiment trails (P < 0.05). In female: RMSSD varied from 46.73 ± 5.34 at before to 46.59 ± 5.84 at during and to 44.74 ± 5.60 at after; there was no statistically significant difference between any two of the three experimental trails (P > 0.05). Therefore, nicotine ingestion had significant effect on reducing male RMSSD, which indicated that the PNS activity of CAM was significantly decreased upon nicotine ingestion in males.RMSSD analysis, in male: RMSSD varied from 58.64 ± 5.70 at before to 57.69 ± 7.27 at during and to 52.38 ± 5.25 at after; there was a statistically significant difference between experimental trails after and before , HF (nu), and HR: there was no any significant difference in % change between male and female in LF (nu), HF (nu), and HR from before to during and to after upon 4 mg nicotine ingestion , HF (nu), LF/HF, HR, and % change in LF (nu) and HF (nu) and HR, they shown that nicotine had effects in reducing HRV, where the CAM activity was shifted to SNS predominant manner rather than PNS, and these effects were not gender different or gender dependent.P < 0.05) (LF (nu), HF (nu), LF/HF, RMSSD, and HR were not statistically significantly different between males and females, while SDNN in male was 93.24 ± 6.50 which was statistically significant higher than female  .P < 0.05), and cause the development of metabolic associated fatty liver disease in adulthood offspring mice [Nicotine can enter the body in many ways, such as smoke inhalation, ingestion, transdermal patch, and intranasal spray. In human, nicotine can easily pass through the blood brain barrier and enter the brain after it enters the human blood circulation, and it takes just 10 to 20 seconds to get nicotine from smoke into the brain. Nicotine has been found to have cognitive-enhancing effects  and can ing mice , 18.In mammals, nicotine binds with nicotinic acetylcholine receptors (nAChRs). nAChRs are composed of five subunits arranged around a water filled pore  and mainIn the heart, nAChRs are expressed on cardiac myocytes. Nicotine binds to cardiac nAChRs will lead to the activation of cardiac nAChRs, resulting in CAM imbalance. CAM imbalance, characterized by a hyperactive SNS and a hypoactive PNS, is associated with various pathological conditions. Over time, excessive energy demands on the system can lead to cardiovascular disease. HRV can be used to assess CAM imbalance; therefore, the risk of cardiovascular disease is commonly found in people with smoking.Many researches have done earlier in smoking subjects found that NRT in smoking cessation have no significant disturbances of autonomic cardiac control  when compared with smoking event in smokers –23. HoweIn this experiment, nicotine effect was investigated in young healthy nonsmokers rather than smokers, the nicotine drug used here was Nicabate CQ lozenge (one of NRT) which contained nicotine at 4 mg dosage. Nicotine at 4 mg dosage was thought to be sufficient to cause an obvious effect despite the weight differences of subjects, because 4 mg nicotine was sufficient for smoking cessation in high-dependence smokers with a big range of weight (~76 ± 18 kg) . In addiLF measurement mainly represents SNS activity, and HF measurement mainly represents PNS activity. Therefore, in this experiment, the results clearly shown that nicotine indeed has an adverse effect on HRV in both healthy males and females  and leads to the transfer of CAM to SNS predominated control (LF/HF increased). The nicotine effect on CAM is rapid as the significance in LF, HF, LF/HF, and HR was observed at only 20 min after nicotine ingestion. Therefore, this experimental results shown that nicotine does have an adverse effect on HRV. HRV is the indicator for predicting the fitness of a person  and usedOverall, nicotine is harmful to cardiac system, and smoking or nicotine replacement therapy (NRT), especially nicotine containment at 4 mg dosage, is not safe for Chinese young adults.


To determine the hypnotic and analgesic effects of brimonidine, and evaluate its efficacy and safety for general anesthesia. Potentiation of pentobarbital sleeping time following brimonidine administration was observed in mice, as was the analgesic activity of brimonidine.50) and lethal dose (LD50) of intraperitoneally injected brimonidine were determined in hypnotized mice. In addition, the LD50 of intravenously injected brimonidine, and ED50 of intravenously, intramuscularly, and intrarectally injected brimonidine in hypnotized rabbits were determined. Finally, the synergistic anesthetic effect of brimonidine and chloral hydrate was evaluated in rabbits.The median effective dose (ED50 of intraperitoneally administered brimonidine in hypnotized mice was 75.7 mg/kg and the LD50 was 379 mg/kg. ED50 values of intravenous, intramuscular, and intrarectal brimonidine for hypnosis in rabbits were 5.2 mg/kg, 8.8 mg/kg, and 8.7 mg/kg, respectively; the LD50 of intravenous brimonidine was 146 mg/kg. Combined intravenous administration of 0.6 mg/kg brimonidine and 0.03 g/kg chloral hydrate had a synergistic anesthetic effect.Intraperitoneal injection of 10 mg/kg brimonidine enhanced the hypnotic effect of a threshold dose of pentobarbital. Intraperitoneally injected brimonidine produced dose-related analgesic effects in mice. The EDBrimonidine elicited hypnotic and analgesic effects after systemic administration and exhibited safety. Moreover, brimonidine enhanced the effects of other types of narcotics when combined.The online version contains supplementary material available at 10.1186/s12871-021-01516-1. General anesthesia involves drugs that widely inhibit the central nervous system to temporarily inhibit consciousness and nerve response activities, such that the body has no response or memory to pain and noxious stimulation. Such effects may be accompanied by a decrease of muscle tension caused by central mechanisms, but the important central functional state of the medulla oblongata is maintained.2 Adrenergic receptor agonists, such as dexmedetomidine, are often used as adjuncts to clinical general anesthesia, but can also be used as a basic anesthetic in combination with local anesthetics for surgery. Such anesthetics act as both sedatives and analgesics, and may have hypnotic, anti-anxiety, anti-sympathetic, and muscle relaxation effects. Moreover, these anesthetics protect organs, have good safety profiles, are not addictive.α2 adrenergic receptor agonists are often used in combination with other anesthetics to meet the anesthesia needs of various surgical settings during the induction period, maintenance period, and awakening period. Dexmedetomidine and clonidine are already widely used for clinical anesthesia. Their characteristics include good hypnotic and analgesic effects, as well as the abilities to strengthen the efficacy of other central sedative and analgesic drugs, and reduce the dosage of other drugs , thus reducing toxicity and side effects. Similar anesthetic effects could be expected using brimonidine.α2 receptor agonist with high activity and good water-fat solubility, and preliminarily evaluated its safety. At present, no reports describe induction or maintenance of general anesthesia by brimonidine alone or in combination. This study evaluated the analgesic and hypnotic potentiation of brimonidine systemic administration against various animal experimental models. Here, we report the first investigation into the general anesthesia activities of brimonidine.In this study, we investigated the general anesthesia effects of brimonidine, a highly selective αJapanese white rabbits of ordinary grade , weighing 2.3–2.6 kg were used in experiments [Animal Production License No. SCXK (Beijing) 2019–0006]. In addition, specific-pathogen-free Kunming (KM) mice , weighing 20.0–23.0 g were used [Animal Production License No. SCXK (Beijing) 2016–0006]. Animal experiments were conducted in the Animal Room of Tianjin Institute of Medical and Pharmaceutical Science. Rabbits were maintained in conventional animal rooms and individually housed in stainless steel cages, while mice were housed in a cleanroom environment in cages. Animals were maintained in air-conditioned rooms with a temperature of 22 °C ± 3 °C, 55% ± 15% relative humidity, and 12-h light/dark cycle. Animals were fed a commercial diet from Keao Xieli Feed  twice daily and given tap water (rabbits) or purified water (mice) ad libitum. Standard procedures and housing conditions were applied in a facility accredited by the Tianjin Municipal Science and Technology Bureau. Animals were anesthetized before euthanasia. Permission and approval for animal studies were obtained from the Animal Ethics Committee of Tianjin Institute of Medical & Pharmaceutical Science .Brimonidine powder (Batch No. AG08-PK20200701) was purchased from Xingkaiyue Biotechnology . Xylazine hydrochloride injection  was purchased from Best Biological Technology Institute . Chloral hydrate powder (Batch No. 2020304) was purchased from Kermel Chemical Reagent . Pentobarbital (Batch No. 127 K1005) was purchased from Huanyu Biotechnology . Methanol  was purchased from Chemical Reagent wholesale company .50 for intraperitoneal anesthesia of mice. For the Brimonidine A group, a 1.1-fold gradient solution series at concentrations of 4.5, 4.9, 5.4, and 6.0 mg/mL was used to determine the ED50 for intravenous anesthesia in rabbits. For the Brimonidine B group, a 1.1-fold gradient solution series at concentrations of 37.5, 41.5, 45.5, and 50.0 mg/mL was used to determine the ED50 for muscular anesthesia in rabbits. A 0.85-fold gradient series of brimonidine solutions at concentrations of 52.1, 44.2, 37.6, and 32.0 mg/mL was used to determine LD50 by intraperitoneal injection in mice. A 0.9-fold gradient series of brimonidine solutions at concentrations of 18.0, 16.4, 14.9, 13.5, and 12.3 mg/mL was used to determine LD50 by intravenous injection in rabbits.Chloral hydrate was prepared as 10% chloral hydrate solution in normal saline. Pentobarbital was prepared as 3 mg/mL and 4 mg/mL solutions in physiological saline. A five-fold gradient brimonidine solution series at concentrations of 0.04, 0.008, and 0.0016 mg/mL was prepared for the writhing test. A 1.2-fold gradient brimonidine solution series at concentrations of 5.5, 6.7, 8.0, and 9.6 mg/mL was prepared to determine the EDTo combine brimonidine and chloral hydrate, brimonidine at a dose of 2 mg/mL was added to a 10% solution of chloral hydrate. To combine brimonidine and pentobarbital, brimonidine at a dose of 1 mg/mL was added to a 3 mg/mL solution of pentobarbital.Mice were randomly divided into experimental, control, and threshold-dose pentobarbital groups . Each animal in the experimental group was intraperitoneally injected with a combination of brimonidine and pentobarbital at a dose of 0.1 mL/10 g, while the control group and threshold-dose pentobarbital group were intraperitoneally injected with 4 mg/mL and 3 mg/mL pentobarbital solution at a dose of 0.1 mL/10 g, respectively. Mice were then placed in an incubator to maintain their body temperature. Whether the righting response disappeared was recorded, as was the time of disappearance and recovery of the righting response. Onset of sleep was the time that animals stayed immobile and lost their righting reflex.Mice were randomly divided into high-dose (0.4 mg/kg), medium-dose (0.08 mg/kg), low-dose (0.016 mg/kg), and control groups, with three males and three females per group. Mice in high-, middle-, and low-dose groups were intraperitoneally injected with 0.04, 0.008, and 0.0016 mg/mL brimonidine solution at a dose of 0.1 mL/10 g, respectively, while mice in the control group were intraperitoneally injected with normal saline at a dose of 0.1 mL/10 g. Mice in the control group were intraperitoneally injected with 0.1 mL/10 g saline. Fifteen minutes after administration of all treatments, mice were intraperitoneally injected with 0.6% acetic acid at a dose of 10 mL/kg. Numbers of writhing responses observed during a 20-min period were counted and recorded. Each writhe, defined as the stretching of the abdomen and/or stretching of at least one hind limb, was recorded with a stopwatch.The percentage of analgesic activity was calculated as follows:Mice were divided into four groups  that were administered 55.6, 66.7, 80.0, or 96.0 mg/kg brimonidine. Mice in each group were intraperitoneally injected with a 1.2-fold gradient brimonidine solution series at a dose of 0.1 mL/10 g. When mice were still in the supine position, they were placed on the heat preservation table in the supine position to maintain their body temperature.Observation indicators: (1) Behavioral and autonomous activities of mice: the appearance, posture, and mental state of mice were observed as activity  and inactivity (prostrate without motion). (2) Ratio of sleeping: the incidence of loss of righting response was observed following drug administration. The index of sleep falling was evaluated as the loss of righting response for more than 1 min. Numbers for each group were observed in mice sleeping within 1 h. (3) Sleeping time: the time from administration to the disappearance of righting response and recovery of righting response was recorded. The time from drug administration to disappearance of the righting response was considered the sleep latency, while the time from disappearance of the righting response to recovery of the righting response was considered the sleep duration.50 by modified Karber methodXm: logarithmic value of the maximum-dose group doseI: Logarithmic ratio of high-dose to low-dose for two adjacent groupsP: Ratio of sleeping of animals in each group, expressed as a decimaln: number of animals in each groupCalculation of EDn = 10): 4.5 mg/kg, 4.9 mg/kg, 5.4 mg/kg, and 6.0 mg/kg. Rabbits in each group were injected with a 1.1-fold gradient solution series of Brimonidine A at a dose of 1.0 mL/kg. When rabbits were inactive, they were required to assume the supine position.Rabbits were divided into four groups (Observation indexes: (1) Behavioral and autonomic activities of rabbits: the appearance, posture, and mental state of rabbits were characterized by when they walked with or without using their legs (considered as activity), while their prostration was considered as quiet. (2) Ratio of sleeping: the incidence of disappearance of acupuncture response after medication was observed. The index of acupuncture sleep falling was calculated as the disappearance of acupuncture response for more than 1 min, while sleep falling numbers were observed for 1 h in each group. (3) Sleeping time: time from the disappearance of acupuncture response to the recovery of acupuncture response was calculated as the sleeping time.50, as described above.The modified Karber method was used to calculate EDn = 10 per group): 7.5 mg/kg, 8.3 mg/kg, 9.1 mg/kg, and 10.0 mg/kg. Rabbits in each group were injected with a 1.1-fold gradient solution series of Brimonidine B at a dose of 0.2 mL/kg. When rabbits were inactive, they were required to assume the supine position.Rabbits were divided into four groups : 7.5 mg/kg, 8.3 mg/kg, 9.1 mg/kg, and 10.0 mg/kg. Rabbits in each group were injected with a 1.1-fold gradient solution series of Brimonidine B at a dose of 0.2 mL/kg.Rabbits were divided into four groups /(n-1)]}0.5, where d is the log difference between two adjacent doses.S50 was measured and calculated as described above for the determination of LD50 in mice.Rabbits were intravenously injected with a 0.9-fold gradient series of brimonidine solutions at a dose of 10 mL/1 kg, one at a time. LDTwenty-four rabbits were divided into three groups (eight rabbits per group): low-dose, high-dose, and control groups. The low-dose group was intravenously injected with 0.3 mL/kg brimonidine and chloral hydrate, while the high-dose group was injected with 0.6 mL/kg brimonidine and chloral hydrate. After intramuscular injection of 0.15 mL/kg xylazine, the control group was injected with 1.5 mL/kg of 10% chloral hydrate at a rate of 2–3 mL/min.50 in rabbits given intravenous injection. After the acupuncture response recovered, rabbits in low-dose and high-dose groups were intravenously injected with 0.15 mL/kg brimonidine and chloral hydrate, and then the re-sleeping time was observed.Behavioral and voluntary activities were recorded from the time of administration to the disappearance and recovery of acupuncture responses. Sleep latency was recorded as the time between pentobarbital administration and sleep onset. Sleeping time was observed as described above for the determination of hypnotic EDn = 3), 6 mg/mL brimonidine was injected to the ear vein. Blood samples were collected from each rabbit at 5, 15, 30, and 45 min; and 1, 2, and 3 h via the heart. Blood samples were cooled on ice immediately after blood sampling, and plasma was obtained by centrifugation (2000×g for 10 min). All bioanalysis samples were stored at − 80 °C until processing. Brimonidine concentrations in plasma samples were determined by high-performance liquid chromatography (HPLC) assay. The plasma samples were pretreated with methanol extraction methods. The extracted samples were analyzed by HPLC-20A . Brimonidine was eluted on a ODS-BP column  using a mobile phase of 15% methanol. Brimonidine was detected at 246 nm. T1/2 and area under the curve (AUC0–∞) in the plasma were determined.For each rabbit .After injection, mice in the experimental groups gradually developed ataxia and decreased exercise. Approximately 3–5 min after injection, mice exhibited a negative rollover response and the ratio of sleeping was 100%, as defined by an increased sleeping time (118.7 ± 4.6 min) in treated mice compared with the control group  and control groups (40 mg/kg), mice developed ataxia, decreased their mobility, and then lay down quietly. However, during the 2-h observation period, the ratio of sleeping in the threshold-dose pentobarbital group was always 0, and no micturition occurred.Following intraperitoneal injection of brimonidine for 15 min, high-dose group mice exhibited decreased activity. The middle-dose group was still active, but there was no courtship action, whereas the low-dose group moved freely and exhibited courtship actions such as raising forelimbs. In the high-dose group (0.4 mg/kg), there was no writhing response within 20 min of intraperitoneal injection of acetic acid solution. In the middle-dose group (0.08 mg/kg), most mice showed writhing reactions  was 70.2–81.6 mg/kg.Hypnotic effects are summarized in Table During half injection, rabbits struggled impatiently for several seconds and then calmed down. During the incubation period, the front legs were straight, the body was shaking, the tail was straight, the pupils became narrowed, the fundus was pale, and the limbs gradually relaxed. During sleep, the rabbit may experience phenomena such as eyeball shaking, micturition, and body surface temperature drop, or may wake up temporarily by changing the body position or twisting the limbs.50 of brimonidine solution was 5.2 mg/kg and the 95% CI was 5.0–5.4 mg/kg.Hypnotic effects were summarized in Table In the 6-mg/kg dose group, all rabbits entered the sleep stage, the induction period was 8.2 ± 3.3 min, and sleep duration was 128.1 ± 16.9 min Fig. . HoweverThere was no restlessness or struggle during intramuscular injection of rabbits. Moreover, the latency response was obviously reduced compared with intravenous injection, the sleep induction period was less than 3 min, and most rabbits did not exhibit muscle tremor or horn arch rebound. During sleep, rabbits exhibited symptoms such as nystagmus, micturition, and temperature drop on the body surface. Changing the body position or twisting the limbs could temporarily wake up the rabbits.50 of intramuscular brimonidine injection was 8.8 mg/kg and the 95% CI was 8.5–9.1 mg/kg.Hypnotic effects are summarized in Table All rabbits in the 10-mg/kg group fell asleep, with an induction period of 6.4 ± 1.8 min and sleeping time of 19.0 ± 11.3 min Fig. .There was no restlessness or struggle during intrarectal injection of rabbits. Neither muscle tremors nor the horn arch rebound was observed.50 of intrarectal brimonidine injection was 8.7 mg/kg and the 95% CI was 8.3–9.1 mg/kg.Hypnotic effects are summarized in Table All rabbits in the 10-mg/kg group fell asleep, with an induction period of 11.0 ± 4.1 min and sleeping time of 53.6 ± 27.9 min . However, there was no difference in sleeping times between low-dose and high-dose groups .The induction periods of low-dose and high-dose groups were shorter than 1 min. After intravenous injection, all animals exhibited miosis, white fundus, turning over, decreased muscle tension, and no limb stiffness. Rabbits in both groups entered the anesthesia period with similar sleeping times. During the anesthesia period, rabbits exhibited nystagmus and decreased body surface temperatures, but did not urinate and could not temporarily wake up by changing the body position or twisting the limbs. In the control group, the induction period was 3.3 ± 1.4 min, mild symptoms (such as limb stiffness) were exhibited, and one death occurred  rabbits which are exhibited in Fig. Brimonidine is clinically used in the treatment of glaucoma and facial congestion. No previous study has investigated the induction or maintenance of general anesthesia with brimonidine; however, a report of brimonidine is used for pre-anesthetic administration for animals, such as intraperitoneal injection of 30 μg/kg brimonidine, diminished the sympathetic nerve effect of ketamine general anesthesia in rats .Many clinical reports indicate that local and systemic administration of brimonidine can cause side effects in the central nervous system, resulting in nervous system symptoms such as sedation, lethargy, and even death . Followi2 receptor agonist action of brimonidine [This study confirmed that brimonidine has good hypnotic, sedative, and analgesic effects on mice and rabbits. This result is related to the αmonidine .2:α1 selectivity coefficient of brimonidine is 1780:1, which is higher than 1620:1 for dexmedetomidine and 220:1 for clonidine [2:I1 selectivity coefficient of brimonidine is 100:1, which is higher than 32:1 for dexmedetomidine and 16:1 for clonidine [2 receptor subtypes in humans, primates, pigs, and rabbits , of which the α2a receptor is involved in general anesthesia. Brimonidine has moderate α2a receptor selectivity for α2 receptor subtypes. The Ki values of dexmedetomidine for α2a, α2b, and α2c receptors are 6.2, 4.0, and 6.0 nM, respectively, whereas these values are 3.7, 512, and 120 nM, respectively, for brimonidine [1, I1, and α2b receptors affects the stability of cardiovascular dynamics [1, I1, and α2b receptors, the side effects of brimonidine are lighter than clonidine and dexmedetomidine [Brimonidine is highly receptor-selective and has more advantages than dexmedetomidine and clonidine. The αtomidine .2a receptors hyperpolarizes nerve cells in the locus coeruleus, which reduces the release of norepinephrine and inhibition of γ-GABA neurons in the ventrolateral anterior cross nucleus of the hypothalamus to induce sedative hypnotic effects. The ED50 of brimonidine for inducing potassium influx in rat locus coeruleus neurons in vitro is 4.6 * 10− 8 M, while 10− 6 M brimonidine can induce the maximum current [The locus coeruleus brainstem is an important functional area in the central nervous system that regulates arousal. Activation of αThe hypnotic assessment method was based on prolongation of sleep induced by pentobarbital, a classic pharmacological method for screening sedative hypnotic agents , 12. The50 values for mouse hypnosis  and rabbit hypnosis , as well as sleeping times of mice  and rabbits . The observed difference in hypnotic doses of brimonidine for mice and rabbits may be related to the difference in species. Thus, if brimonidine is developed as an anesthetic for experimental animals, it is more suitable to be used alone in rabbits than mice.In this study, we determined ED2 receptor agonist can exert analgesic and noxious stimulation inhibitory effects in peripheral nerves, spinal cord, and upper spinal cord [2a subtype is contained in the spinal dorsal horn and primary sensory neurons, whereby adrenergic receptor agonists bind this receptor to trigger postsynaptic inhibition of secondary afferent neurons and presynaptic inhibition of primary sensory neurons, thus playing a major role in inhibiting pain signals [The αnal cord . The α2a signals .50. Compared with 0.016 mg/kg brimonidine, 0.08 mg/kg brimonidine had a sedative effect and an increased analgesic effect. A dose of 0.4 mg/kg brimonidine decreased the activity of mice, and no acetic acid-induced peritoneal pain reaction was observed. These results indicate that brimonidine significantly reduced the number of writhes in a dose-dependent manner compared with the negative control. However, because sedative and analgesic effects are sometimes difficult to distinguish, especially for elevated concentrations, it remains uncertain whether the disappearance of pain responses in high-dose group mice was entirely due to the analgesic activity of the drug or partly affected by sedation.The acetic acid-induced writhing reflex mouse model is a widely accepted, simple, sensitive, and effective pain model for evaluating peripherally acting analgesics. The pain caused by acetic acid liberates endogenous substance to elicit peripheral- and central-mediated analgesic actions . In the 50 of 0.37 nM, and the combination of brimonidine with an epidural (i.e. opioid) led to synergistic analgesia [Previous studies reported that brimonidine had good independent analgesic effects. For example, inhibition of thermal nociception in mice following epidural administration of brimonidine had an analgesic EDnalgesia .2 receptor agonists can theoretically cause presynaptic inhibition of motor neurons and play a certain role in muscle relaxation [The hypnotic effect of brimonidine was more obvious in rabbits than in mice. Our experiments found that intravenous administration of 4.5 mg/mL brimonidine could make the righting response disappear. During this period, the limbs could be placed at will, but pinching the limbs could cause muscles of the limbs to contract. Although αlaxation , muscle 2 receptors in the paraventricular nucleus of the hypothalamus, thereby inhibiting the release of antidiuretic hormone to cause an increase in urine output. Simultaneously, brimonidine inhibited norepinephrine release and contraction of the bladder sphincter, leading animals to urinate. Dexmedetomidine had a similar effect on diuresis in clinical applications [Although brimonidine has hypnotic and analgesic effects because it indirectly inhibits the ascending activation system of the reticular structure through the locus coeruleus, it does not block the awakening mechanism while accompanying sleep . We founications . This is2 receptor agonists can activate dopaminergic neurons in the ventral tegmental area, which increases dopamine concentrations in the related forebrain projection areas [After intravenous administration of a high dose of brimonidine, the hypnotic effect was weakened, not enhanced. In previous experiments, a large portion of animals may not have been fully anesthetized when the intravenous drug dose was more than 6.7 mg/kg or intrarectal dose was more than 12.3 mg/kg. During induction of general anesthesia, high-dose αon areas . During When judging the anesthetic effect of brimonidine alone or in combination with chloral hydrate by various routes of administration, the recommended dose-selection principle  is to use the minimum amount of drug that achieves a sufficient anesthetic effect in all animals with minimal side effects.2 receptor agonists such as brimonidine can lead to hypothermia [To overcome these shortcomings and solve potential problems with future application, the combination of brimonidine with other anesthetics is a good method. The results of experiments examining the hypnotic effect of brimonidine on increasing the threshold dose of pentobarbital confirmed the hypnotic effect of brimonidine. Moreover, these results revealed that 10 mg/kg brimonidine combined with 30 mg/kg pentobarbital hypnotized all mice for more than 2 h after intraperitoneal injection, without any death phenomenon, which is beneficial for long-term operations of mice. However, αothermia , meaningIn addition to pentobarbital, a combination of brimonidine and chloral hydrate was also found to have a good synergistic effect. When brimonidine and chloral hydrate were used together, the doses of the two drugs in the low-dose group were only one-tenth of the original single dose. In addition, the low-dose group exhibited rapid onset of anesthesia, a stable anesthesia effect, and convenient supplement of anesthetics. Although the dosage of the high-dose group was double that of the low-dose group, the anesthetic effect was similar. Thus, combining brimonidine and chloral hydrate has the potential advantages of stable effects and high cost performance for animal anesthesia.50 of brimonidine. Moreover, no deaths occurred when brimonidine was administered in combination with pentobarbital or chloral hydrate.In terms of safety, the therapeutic index of intraperitoneal brimonidine administration in mice was 5.0, while the therapeutic index of intravenous brimonidine in rabbits was 28.0, indicating that the safety range of brimonidine was wider in rabbits than mice. In this study, no mortality occurred during experiments using the EDMany reports have indicated the safety of brimonidine administered to animals at high doses; for example, oral administration of brimonidine to rats for up to 1 year (2.5 mg/kg/day) did not cause organ toxicity . In a stP < 0.05). Use of brimonidine in clinical eye drops as a drug for glaucoma yielded high local absorption without local tissue irritation because of its physical properties. First, the pKa value of brimonidine is 7.78, yielding an aqueous solution with an approximately neutral pH. Second, the logP value of brimonidine is 0.83, which is similar to the logP of clonidine (0.73) used for mucosally administered general sedation [Sleeping times of rabbits in the 10-mg/kg intramuscular anesthesia group were shorter than observed in the 10-mg/kg intrarectal anesthesia group  of brimonidine potentiates pentobarbital hypnosis in mice.Additional file 2: Table 2. Number of writhes induced by acetic acid in mice.Additional file 3: Table 3. Hypnotic effects of intraperitoneal brimonidine in mice.Additional file 4: Table 4. Hypnotic effects of intravenous brimonidine in rabbits.Additional file 5: Table 5. Hypnotic effects of intramuscular brimonidine in rabbits.Additional file 6: Table 6. Hypnotic effects of intrarectal brimonidine in rabbits.Additional file 7: Table 7. LD50 of brimonidine in mice evaluated with up-and-down sequential method.Additional file 8: Table 8. LD50 of brimonidine in rabbits evaluated with up-and-down sequential method.Additional file 9: Table 9. Synergy of hypnotic effects of brimonidine combined with chloral hydrate.Additional file 10: Table 10. Brimonidine concentrations in the plasma after the intravenous administration (mg/ml).


Metal implants are used worldwide, with millions of nails, plates, and fixtures grafted during orthopedic surgeries. Iron is the most common element of these metal implants. As time passes, implants can be corroded and iron can be released. Ionized iron permeates the surrounding tissues and enters circulation; importantly, iron ions pass through the blood–brain barrier. Can iron from implants represent a risk factor for neurological diseases? This remains an unanswered question. In this study, we discovered that patients with metal implants delivered through orthopedic surgeries have higher incidence of Parkinson’s disease or ischemic stroke compared to patients who underwent similar surgeries but did not have implants. Concentration of serum iron and ferritin was increased in subjects with metal implants. In experiments in vivo, we found that injection of iron dextran selectively decreased the presence of divalent metal transporter 1 (DMT1) in neurons through increasing the expression of Ndfip1, which degrades DMT1 and does not exist in glial cells. At the same time, excess of iron increased expression of DMT1 in astrocytes and microglial cells and triggered reactive astrogliosis and microgliosis. Facing the attack of excess iron, glial cells act as neuroprotectors to accumulate more extracellular iron by upregulating DMT1, whereas neurons limit iron uptake through increasing DMT1 degradation. Cerebral accumulation of iron in animals is associated with impaired cognition, locomotion, and mood. Excess iron from surgical implants thus can affect neural cells and may be regarded as a risk factor for neurodegeneration.  At present, the follow-up mainly focuses on the operative prognosis, functional recovery, complications, and secondary pathologies. Effects of metal implants on the nervous system and neurological diseases, however, are rarely considered. Metal implants are mainly composed of pure iron or iron-based alloys, the latter being more widely adopted in clinical practice. The common metal alloys contain Mn, Ti, Mg, Al, Co, Si, etc., yet iron remains the primary element.3+-bound to transferrin (holo-TF), or, for non-TF-bound iron, by membrane transport of Fe2+ mediated by divalent metal ion transporter 1 (DMT1/SLC11A2), which underlies Fe2+ uptake through the plasma membrane or from endosomes.,+-driven multimetal transporter responsible for transmembrane transport of nontransferrin-bound iron.Ionized iron crosses blood–brain barrier (BBB) and blood–cerebrospinal barrier.In this study, we performed retrospective analyses of patients with the history of metal implants; in particular, we focused on patients diagnosed with PD, ischemic stroke, or neurologic tumors (a disease-related control group). We discovered that metal implantation increased the occurrence of PD and ischemic stroke. Subsequently, we compared the PD occurrence in the patients subjected to orthopedic surgeries with and without metal implants. In addition, we measured the levels of serum iron, ferritin, and transferrin in the healthy control subjects and in patients who underwent surgical removal of implants immediately prior to the surgery. In the animal model, we studied how exogenous iron acts on neural cells, and we found the opposite effects of iron on the levels of DMT1 in neurons and glia. Glial cells respond to exogenous iron attack by upregulating DMT1 expression, whereas neurons accelerated DMT1 degradation; both these processes being arguably neuroprotective. Our study suggests that iron intake associated with orthopedic implants should be considered as a risk factor for neurological disorders.n = 50), and these metal implants were iron-based alloys grafted to patients with fracture of femoral shaft. The healthy subjects were the age-matched individuals who did not have neurological diseases and the history of surgeries. The anamneses were collected from the First Affiliated Hospital of China Medical University, the biggest general public hospital in the northeast of China with about 3 million outpatients and 159 000 inpatients every year. We selected cases of inpatients with definitive diagnosis from 2009 to 2018. Population sources were from the four provinces of China . The individuals in healthy control group were similarly selected from these four provinces. This study was authorized and approved by Medical Ethics Committee of China Medical University, No. [2019]059.First, we set up a case-control study with common cerebral diseases, including 500 patients diagnosed with PD, 500 cases of ischemic stroke, 200 cases of brain tumor , and 700 healthy controls. Patients with tumors were used as the disease-related control group. Then, for investigating the effects of orthopedic surgeries with and without metal implants on the risk of PD, we established a case-cohort study with the inpatients from orthopedic departments, including 15 000 patients with metal implant surgeries and 7500 patients who underwent orthopedic surgeries without implantation. To further analyze the effects of metal implants, blood samples were collected from 50 inpatients before surgical removal of metal implants and 50 healthy controls, all these participants gave their written informed consent to take part in this study. Both groups have the similar average age and the same gender ratio, 30 male and 20 female. The average holding time of metal implants before removal surgery was 18.98 ± 2.40 months  ages more than 18 years, (2) PD diagnosis: patients were diagnosed according to UK PD Society Brain Bank Clinical Diagnostic,Under the permission of patients and the management regulations of operating rooms, metal implants removed from the patients were inspected and images of the surface were taken by the video microscope .TM II fully automatic analyzer .Serum iron was measured by Sysmex xs-800i Automatic Hematology Analyzer . Serum ferritin was detected with electrochemiluminescence technology and measured by Roche ELECSYS 2010 analyzer . Transferrin was determined with an immunonephelometric assay and a SIEMENS BNMale wild type mice, as well as B6.Cg-Tg(Thy1-YFP)HJrs/J and FVB/N-Tg(GFAP-GFP)14Mes/J strains were obtained from Jackson Laboratory . Mice were 10–12 weeks old (∼25 g) and were kept in standard housing conditions  with food and water available ad libitum. All operations were carried out in accordance with the USA National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023) and its 1978 revision, and all experimental protocols were registered in and approved by the Institutional Animal Care and Use Committee of China Medical University.TM 555 donkey anti-Rabbit (Cat#: A31572) and Alexa FluorTM 488 donkey antimouse (Cat#: A21202) was purchased from Thermo Fisher Scientific . Primary antibody raised against Iba-1 (Cat#: 019-19741) was purchased from Wako Pure Chemical Industries . Micro serum iron concentration assay kit (Cat#: BC1735), potassium ferrocyanide, and diaminobenzidine (DAB) kit (Cat#: DA1015) were purchased from Solarbio Life Sciences . TUNEL apoptosis detection kit (Cat#: 11772457001) was purchased from Roche-Sigma (USA). The ROS assay kit (Cat#: BB-470532) was purchased from BestBio . The quantitative PCR  was purchased from TaKaRa Bio .Most chemicals, including iron dextran  and 4′, 6-diamidine-2′-phenylinedole dihydrochloride  were purchased from Sigma . The primary antibody raised against DMT1 (Cat#: sc-166884) was purchased from Santa Cruz Biotechnology . Primary antibody raised against NeuN (Cat#: PA5-78639), GFAP (Cat#: PA5-78639) and secondary antibodies Alexa FluorIron dextran was dissolved in water at the weight ratio of 1:3 (iron/dextran), the solution of iron dextran was hypodermically injected 200 µL with iron content 1, 2, or 4 mg/kg/day for 6 days, once every day, the same concentration of dextran without iron in the same volume was injected to the control group.,Brain tissue was immersed in 4% paraformaldehyde and cut into 50 μm slices. As described previously,Tissue iron accumulation was visualized by Perl’s staining with modifications. The slices were immersed in the mixed reaction liquid (4% potassium ferrocyanide: 4% hydrochloric acid = 1:1) at 37°C for 30 min and then stained using a DAB kit for 4 min. The slices were imaged using an optical microscope . The iron accumulation was calculated by the ratio of the Perl’s stained area and the total area.The blood samples were centrifuged at 3000 r/min for 20 min to collect the supernatant. The concentration of serum iron was measured with an assay kit and the operations were followed the manufacturer’s protocols. The absorption at 520 nm was measured using ELX808 microplate reader . The iron concentration was calculated by the curve of standard substance.,To measure the mRNA of DMT1 and Ndfip1, we sorted the cells labeled by fluorescent markers YFP for neurons (Thy1-YFP transgenic mice) and GFP for astrocytes (GFAP-GFP transgenic mice). As described previously,,Total RNA was reverse transcribed and PCR amplification was performed in a Robo-cycler thermocycler, as our previous description.As described previously,As in our previous description,The pole test for assessing movement abnormalities was performed as previously described.To assess motor coordination in mice, a rotarod test was performed as previously described.Open field test for assessing anxiety behavior was conducted with an open field box (60 × 60 × 40 cm) which was divided into 9 squares, as previously described.To evaluate neuronal apoptosis in four different regions, TdT-mediated dUTP-biotin nick end labeling (TUNEL) in conjunction with immunofluorescent staining for NeuN was performed as previously described.The tissues from frontal cortex (FC), HP, SN, and ST were divided and centrifuged ×1000 g at 4°C for 10 min. The supernatant was remained and the protein concentration was determined. As per the manufacturer’s protocols of ROS assay kit, adding 10 µL of supernatant and 190 µL of BBoxi Probe O12 detector to a 96-well plate, which was incubated for 30 min at 37°C in dark. Then fluorescence intensity was measured by fluorescence microplate reader . The detection result was presented as the ratio of fluorescence value/mg protein.P < 0.05. For the case-control study, unpaired two-tailed Student’s t-test and X2 test or Fisher exact test (when expected frequencies of the cells of a contingency table <5 were over 20%) were used for analyzing different kinds of data, respectively. For animal experiments, analysis of variance followed by Fisher’s least significant difference or a Tukey–Kramer post hoc multiple comparison test for unequal replications was used for statistical analysis.We used GraphPad Prism 5 software  and SPSS 24 software  for the data statistical analysis, the level of significance was set at The data that support the findings of this study are available from the corresponding author upon reasonable request.X2(1)=8.307, P = 0.004 in PD group, and 5.00% (X2(1)=16.225, P < 0.0001) in ischemic stroke group, while it was only 1.14% in healthy control group. In the cerebral tumor group, the insert incidence was 1.50% (P = 0.716) which was not significantly different from healthy control group.X2(1)=4.414, P = 0.036), in patients older than 75 years, 3 out of 38 underwent metal implantation (P = 0.043). In ischemia group, the inserts frequencies were significantly different from healthy subjects in three age groups: 5 out of 111 in 46- to 60-years-old group (P = 0.024), 12 out of 259 in 61- to 75-years-old group (P = 0.029) and 8 out of 111 in patients older than 75 years (P = 0.016). The distribution of gender, ethnicity, and education showed no difference between all groups. Patients were also divided into three groups, according to the time between implant surgery to PD or ischemia diagnosis, <5, 5–10, and >10 years. The first group showed more cases with metal inserts diagnosed with PD or ischemia (P = 0.002) and 13 out of 25 , respectively.Detailed distribution of clinical cases with inserts was further analyzed with the results shown in ischemia , the incX2(1)=13.143, P < 0.001).In X2(1)=4.132, P = 0.042). In 2010, the PD occurrences in control and inserts groups were 11 out of 530 and 37 out of 893, respectively (X2(1)=4.363, P = 0.037). In 2011, the PD occurrence in these two groups was 10 out of 656 and 43 out of 1418 (X2(1)=4.096, P = 0.043). In the ensuing 7 years, there was no significant difference in the occurrence of PD between control and inserts groups.The yearly numbers of PD-diagnosed subjects and the total number of orthopedic surgeries with or without metal inserts are shown in n = 7500) and 210 ± 3.08 mL , there was no significant difference (P = 0.0544). The anesthesia times were 1.97 ± 0.04 h  and 2.07 ± 0.03 h  in surgeries without and with implants groups, respectively; again, there was no significant difference . Surgeries with metal implants were represented by internal fixation of the distal radius fracture (n = 377). The PD occurrences were 1 out of 199 in the group without implants and 12 out of 377 in the group with implants (X2(1)=4.242, P = 0.039). In lumber spine, lumbar intervertebral disc nucleus pulposus resection and the posterior approach of bone graft fusion with screw fixation system for lumbar spine fracture, PD occurrence was 4 out of 512 and 23 out of 995 (X2(1)=4.499, P = 0.034). On lower limbs, for treating knee osteoarthritis, arthroscopic arthrodesis of the knee and total knee arthroplasty were performed. For these two surgeries, PD occurrence was, respectively, 6 out of 655 and 26 out of 1122 (X2(1)=4.592, P = 0.032).We also compared the occurrence of PD in orthopedic surgeries with or without metal implants in upper limbs, spine, and lower limbs . In uppeP < 0.001, n = 30), in females: 18.93 ± 5.57 µM in control group and 24.19 ± 6.22 µM in implants group , while in females ferritin was 148.46 ± 48.37 µg/L in control group and 274.41 ± 72.84 µg/L in implants group , while in females transferrin was 247.37 ± 38.48 mg/dL in control group and 284.39 ± 40.70 mg/dL in implants group .To detect the effects of implanted iron on the level of iron in body fluids, we collected the blood samples of healthy subjects and patients who experienced metal implants removal surgeries. These patients carried the implants for ∼1.5 years before removal. The representative X-ray images before and after removal surgeries are shown in P = 0.023, n = 6) at 1 mg/kg, 212.89 ± 43.11%  at 2 mg/kg, and 290.22 ± 44.89%  at 4 mg/kg. Four cerebral regions, namely FC, HP, SN, and ST showed iron accumulation following 2 mg/kg iron injections  in FC, 315.08 ± 41.53%  in HP, 202.86 ± 25.53%  in SN and 546.33 ± 37.55%  in ST  of the control group in FC  of the control group in SN  in ST . The exp6) in ST . Iron inup in FC , and to up in SN .P < 0.001; n = 6) of the control group. Administration of iron dextran upregulated mRNA expression of DMT1 to 291.49 ± 62.54%  and 453.13 ± 114.82%  of control groups in astrocytes and microglial cells, respectively. However, iron treatment did not change mRNA expression of Ndfip1 in glial cells.The Nedd4 family interacting protein 1 (Ndfip1) is known to regulate ubiquitination and degradation of DMT1 [19], being thus a leading factor defining transporter presence and operation. Expression of mRNA for DMT1 and Ndfip1 was determined in freshly isolated neurons, astrocytes, and microglial cells . In FACSP < 0.001, n = 6), whereas it increased to 143.92 ± 24.96%  and to 195.30 ± 21.65%  in astrocytes and microglia , 54.74 ± 9.55% , and 46.24 ± 15.87%  in HP, SN, and ST, respectively. In these three brain regions, the intensity of DMT1 was increased to 162.57 ± 27.21% , 169.54 ± 13.64% , and 189.94 ± 53.82%  in astrocytes, and it was elevated to 223.58 ± 29.76% , 142.82 ± 14.72% , and 127.62 ± 15.92%  in microglia, compared with control group , 108.71 ± 6.83% , 121.16 ± 3.67% , 115.03 ± 12.1% , 115.96 ± 5.04% , 112.42 ± 2.89% , 110.93 ± 5.46%  in FC, HP, SN, and ST, respectively  in FC, and to 174.11 ± 57.26%  in SN  in SN .P < 0.001, n = 6), 241.94 ± 65.70% , and 237.75 ± 47.99%  of control group in FC, HP, and SN; there was no significant difference between control and iron dextran groups in ST region  in FC, to 171.39 ± 38.58%  in HP, to 164.01 ± 40.22%  in SN, when compared to control group  was elevated to 243.45 ± 51.59% (T region . Treatmeol group . Howevern = 6) at Day 6. Specifically, the eyes were glazed and sunken, and the color of ears darkened  and 38.97 ± 23.96% , compared with control group , iron decreased neuronal DMT1 protein content, whereas protein levels of DMT1 were increased in astrocytes and in microglial cells. We contemplated that this phenomenon reflects a neuroprotective mechanism activated by iron attack. As summarised in Chronic treatment with iron elevated ROS production in the FC, HP, and SN, but not in the ST. Neuronal apoptosis induced by iron and mediated by ROS was similarly identified in these three regions. Behavioral tests demonstrated that treatment with iron evoked anxiety behaviors and resulted in cognitive and movement dysfunction. Dopaminergic neurons are known to be particularly vulnerable to oxidative damageIn this study, because of the limitation of research time and recorded information, we could not further compare the PD occurrence in patients with metal implants according to the different metal components of implants, the plates coating, their weight, or production origin. The PD occurrence was larger during 2009–2011 period . That maIn conclusion, neuronal apoptosis and reactive gliosis accompany iron overload which promotes neuropathology. The excessive iron should be of special concern and the patients taking iron-based metal implants in orthopedic surgeries have to be considered at risk of the neurodegenerative diseases.APS Function online.This study was supported by Grant No. 81871852 to B.L. from the National Natural Science Foundation of China, Grant No. XLYC1807137 to B.L. from LiaoNing Revitalization Talents Program, Grant No. 20151098 to B.L. from the Scientific Research Foundation for Overseas Scholars of the Education Ministry of China, and Grant No. 202078 to B.L. from Liaoning BaiQianWan Talents Program. Grant No. 81200935 to M.X. from the National Natural Science Foundation of China, Grant No. 20170541030 to M.X. from the Natural Science Foundation of Liaoning Province. Grant No. 81671862 and No. 81871529 to D.G. from the National Natural Science Foundation of China.M.X., A.V., D.G., and B.L. designed and supervised the study; M.X., Shuai Li (S.L.), C.D., Be.C., Bi.C., M.J., Y.Z., and W.G. collected the clinic messages and analyzed the relevant data; Sha.L., G.W., X.Z., D.Z., X.L., and M.Z. performed the experiments in vivo and analyzed the data; B.L. and A.V. wrote the manuscript.The authors have no conflicts of interest to disclose.zqab003_Supplementary_DataClick here for additional data file.