BACKGROUND:Previous studies have shown that human beta-defensin 3 has significant antifungal,antibacterial,and antiviral activities and plays an important bridging role in linking innate and acquired immune responses. OBJECTIVE:To observe the effect of human beta-defensin 3 hydrogel on treatment of periodontitis in rats. METHODS:Using Poloxamer 188 and 407 as the matrix,a blank hydrogel was constructed by cold solution.Human beta-defensin 3 hydrogel was prepared by mixing human beta-defensin 3 with the hydrogel.Twenty-five SD rats were randomly divided into five groups with five rats in each group:No treatment was given in the healthy group.The periodontitis model was constructed by the orthodontic ligature wire method in the periodontitis group,blank hydrogel group,minocycline hydrochloride group,and human beta-defensin 3 hydrogel group.8 weeks after modeling,blank hydrogel,minocycline hydrochloride,and human β-defensin 3 hydrogel were injected into the buccal and palatal periodontal bags,once a week,and relevant tests were carried out after continuous administration for 4 weeks. RESULTS AND CONCLUSION:(1)Compared with the healthy group,periodontal plaque index,gingival bleeding index,and periodontal probing depth were increased in the periodontitis group(P<0.01).Compared with the periodontitis group,the periodontal plaque index,gingival bleeding index,and periodontal probing depth of rats were decreased in the minocycline hydrochloride group and the human beta-defensin 3 hydrogel group(P<0.05).(2)Hematoxylin-eosin staining proved that the hydrogel was not toxic to the rat organism.(3)Stereomicroscopy and Micro CT showed that compared with the healthy group,the root exposure and the distance between enamel cementum boundary and alveolar crest of the periodontitis group were increased(P<0.05).Compared with the periodontitis group,the root exposure and the distance between enamel cementum boundary and alveolar crest of rats were reduced in the minocycline hydrochloride group and human beta-defensin 3 hydrogel group(P<0.05).(4)Hematoxylin-eosin,Masson,and tartrate-resistant acid phosphatase staining showed that periodontal inflammation was obvious,fiber structure was disordered and osteoclasts were active in the periodontitis group and blank hydrogel group,while periodontal inflammation was decreased,fiber arrangement was more regular,and osteoclasts were reduced in the minocycline hydrochloride group and human beta-defensin 3 hydrogel group.(5)qRT-PCR showed that compared with the healthy group,the mRNA expressions of interleukin 1β,tumor necrosis factor α,interleukin 6,and inducible nitric oxide synthase were increased in the periodontitis group(P<0.05).Compared with the periodontitis group,the mRNA expressions of interleukin 1β,tumor necrosis factor α,interleukin 6,and inducible nitric oxide synthase in gingival tissue of rats were decreased in the minocycline hydrochloride group and human beta-defensin 3 hydrogel group(P<0.05).(6)The results showed that human beta-defensin 3 hydrogel was able to attenuate inflammation in rat periodontal tissues by decreasing the relative expression of inflammatory factors and inhibiting osteoblasts.

Objective:To develop and validate a liquid chromatography-tandem mass spectrometry method for determining levofloxacin in plasma sample from pediatric patients.Method:This is a prospective, observational study. The clinical residual plasma samples from healthy individuals for physical examination in Children's Hospital Affiliated to Zhengzhou University were collected as blank matrix. Plasma samples from five pediatric patients who did not receive levofloxacin or ciprofloxacin in the department of Respiration were collected for methodological evaluation. In addition, 34 clinical plasma samples from 22 pediatric patients (9 males and 13 females; mean age (8.1±3.7) years) using levofloxacin was collected, and their plasma concentrations were determined. Using ciprofloxacin as the internal standard, levofloxacin in plasma samples was quantified by liquid chromatography-tandem mass spectrometry following protein precipitation using acetonitrile. A C18 column (Shim-pac GIST-HP C18, 2.1 mm×100 mm, 3 μm) and mobile phase composed of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) with gradient elution at a flow rate of 0.4 ml/min were used to separate levofloxacin. The column temperature was 40 ℃, injection volume was 1 μl and the total analysis time was 9 min. Levofloxacin and ciprofloxacin were ionized with an ESI source in positive ion mode and detected in multiple reaction monitoring (MRM) mode. The detected ions of levofloxacin and ciprofloxacin were m/z 362.10→318.1 and 332.15→231.05, respectively. The method′s specificity, sensitivity, linearity, precision, accuracy, recovery rate, stability, matrix effect, and carry-over were validated. All statistical analyses were performed with SPSS statistical software (version 17.0). The normality of the data was detected by the K-S test. A P<0.05 was considered statistically significant for two tailed tests. Results:The LC-MS/MS method showed a good linearity within the range of 0.062 5-20 mg/L, with the lower detection limit of levofloxacin of 0.062 5 mg/L. The calibration curve for levofloxacin was Y=0.093X+0.010 ( R2>0.99). Under different quality control levels, the accuracy ranged from 92.57% to 104.39%, and the intra-day and inter-day imprecision ranged from 2.32% to 9.35%. These values were not affected by the normal matrix, 5% hemolysis matrix and 15% hyperlipidemia matrix. Furthermore, the levofloxacin plasma samples were stable in the short term. A total of 34 plasma samples from 22 patients were collected and analyzed. Only 2 plasma samples were below the lower limit of quantification, while the other plasma concentrations of levofloxacin were ranged from 0.091 to 6.755 mg/L. Cmax was (5.52 ± 1.09) mg/L. Conclusion:The LC-MS/MS method meets the requirements of the reference method and requires a small sample size (50 μl), making it suitable for the determination of levofloxacin in plasma from pediatric patients.

Polygala tenuifolia,commonly known as Yuanzhi(YZ)in Chinese,has been shown to possess anti-insomnia properties.However,the material basis and the mechanism underlying its sedative-hypnotic effects remain unclear.Herein,we investigated the active components and neurochemical mechanism of YZ extracts using liquid chromatography tandem mass spectrometry(LC-MS/MS)-based pharmaco-metabolomics and mass spectrometry imaging(MSI)-based spatial resolved metabolomics.According to the results,17 prototypes out of 101 ingredients in the YZ extract were detected in both the plasma and brain,which might be the major components contributing to the sedative-hypnotic effects.Network pharmacology analysis revealed that these prototypes may exert their effects through neuroactive ligand-receptor interaction,serotonergic synapse,dopaminergic synapse,and dopaminergic synapse,among other pathways.LC-MS/MS-based targeted metabolomics and Western blot(WB)revealed that tryptophan-serotonin-melatonin(Trp-5-HT-Mel)and tyrosine-norepinephrine-adrenaline(Tyr-Ne-Ad)are the key regulated pathways.Dopa decarboxylase(DDC)upregulation and phenylethanolamine N-methyltransferase(PNMT)downregulation further confirmed these pathways.Furthermore,MSI-based spatially resolved metabolomics revealed notable alterations in 5-HT in the pineal gland(PG),and Ad in the brainstem,including the middle brain(MB),pons(PN),and hypothalamus(HY).In summary,this study illustrates the efficacy of an integrated multidimensional metabolomics approach in unraveling the sedative-hypnotic effects and neurochemical mechanisms of a Chinese herbal medicine,YZ.

OBJECTIVES: To investigate the therapeutic effect of Exocarpium Citri Grandis formula granules (ECGFG) on fatty liver disease (FLD) in zebrafish and explore the underlying mechanism. METHODS: Nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (ALD) models were established in zebrafish larvae at 3 days post fertilization (dpf), in which the treatment efficacy of 16, 32, or 64 μg/mL ECGFG was evaluated by examining zebrafish survival and liver pathologies and using whole-fish oil red O staining and RT-qPCR. The therapeutic mechanism of ECGFG for FLD was investigated using Prussian blue staining, DCFH-DA probe, MDA content detection, RT-qPCR assay and immunohistochemical staining for CAV1. RESULTS: In zebrafish models of NAFLD and ALD, treatment with ECGFG significantly reduced lipid accumulation and the expression levels of FASN, SREBP1, HMGCRA, TNF-α and IL-6, increased the expressions of Apoa1 and PPARα, and reduced iron deposition and the contents of MDA and ROS in the liver. In zebrafish models of NAFLD, treatment with ECGFG at the 3 doses significantly increased hepatic expressions of Tf, TfR, FPN and SLC7A11, and at the doses of 32 and 64 μg/mL, ECGFG obviously increased hepatic expression of GPX4. ALD fish models showed significantly increased hepatic expressions of Tf, TfR and FPN, which were effectively lowered by treatment with ECGFG at the 3 doses. ECGFG did not obviously affect the expression of SLC7A11, but its high dose (64 μg/mL) caused significant elevation of GPX4 expression. Both zebrafish models of NAFLD and ALD showed obviously increased CAV1 expression level in the liver, which was significantly reduced by treatment with 32 and 64 μg/mL ECGFG. CONCLUSIONS In zebrafish models of NAFLD and ALD, ECGFG can alleviate lipid accumulation and inflammatory response and lower the expression level of CAV1 to restore iron homeostasis and suppress lipid peroxidation and ferroptosis in the liver.
Animals ; Zebrafish ; Ferroptosis/drug effects* ; Non-alcoholic Fatty Liver Disease/drug therapy* ; Iron/metabolism* ; Disease Models, Animal ; Lipid Peroxidation/drug effects* ; Homeostasis ; Fatty Liver/drug therapy* ; Liver/metabolism* ; Lipid Metabolism/drug effects* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE:To improve HPLC for content determination of ketoprofen in Ketoprofen enteric-coated capsules. METHODS:HPLC method was adopted. The determination was performed on Chiralpak IC column with mobile phase consisted of n-hexane (0.1% TFA)-isopropanol(90:10,V/V)at a flow rate of 0.8 mL/min. The detection wavelength was set at 268 nm,and column temperature was 25 ℃. The sample size was 10 μL. RESULTS:The linear range of ketoprofen were 0.025-0.5 mg/mL(r=0.9998). The limit of quantitation was 1.0 mg/L,and limit of detection was 0.2 mg/L. RSDs of precision,stability and reproduc-ibility tests were lower than 2%;recoveries were 96.36%-100.32%(RSD=1.87%,n=6). CONCLUSIONS:The method is sim-ple,accurate and rapid,and can be used for the content determination of ketoprofen in Ketoprofen enteric-coated capsules.