ObjectiveTo observe the clinical efficacy and safety of Zhinao capsules in the treatment of mild and moderate sleep disorders in Alzheimer's disease patients with the syndrome of spleen-kidney deficiency and combined phlegm and stasis. MethodsA randomized， double-blind， placebo， controlled clinical trial design was adopted， and 60 Alzheimer's disease patients aged 50-85 years with mild and moderate sleep disorders due to spleen-kidney deficiency and combined phlegm and stasis were included. According to the randomized numble table method， the patients were assigned into a treatment group and a control group （30 in each group， with 29 patients in the treatment group and 28 patients in the control group finally completing the trial）. The treatment group was treated with donepezil hydrochloride tablets combined with Zhinao capsules， and the control group was treated with donepezil hydrochloride tablets combined with the simulant of Zhinao capsules for 12 weeks. The Pittsburgh sleep quality index （PSQI）， smart bracelet sleep monitoring， epworth sleeping scale （ESS）， Mini-Mental State Examination （MMSE）， Activities of Daily Living （ADL）， and traditional Chinese medicine symptoms were scored. Furthermore， the brain-derived neurotrophic factor （BDNF） and gamma-aminobutyric acid （GABA） levels were measured. ResultsAfter 12 weeks of treatment， the total response rate of clinical efficacy assessment in the treatment group reached 89.7%， which was significantly higher than that （21.4%） in the control group （Z=-5.14， P<0.01）. After treatment， the TCM symptom scores declined in both groups （P<0.01）， and the scores in the treatment group were lower than those in the control group （P<0.01）. After treatment， the treatment group showed decreased total score and seven component scores of the PSQI scale （P<0.05， P<0.01）， and the scores in the treatment group were lower than those in the control group （P<0.05， P<0.01）. After treatment， the treatment group showed increases in the duration of deep sleep， light sleep， rapid eye movement sleep， and total sleep at night and decreases in the time to fall asleep， the number of awakenings from sleep at night， and the time for sporadic daytime naps （P<0.01）. The control group did not show significant changes in the duration of light sleep， time to fall asleep， and number of awakenings from sleep at night. Moreover， the treatment group outperformed the control group in terms of the indicators above （P<0.05， P<0.01）. After treatment， the treatment group showed decreased scores of ESS， ADL and its subscales physical self-maintenance scale （PSMS）， and Instrumental Activities of daily living scale （IADLs） and increased score of MMSE and levels of BDNF and GABA （P<0.05， P<0.01）. There was no significant difference in PSMS between the two groups， and the treatment group was significantly better than the control group in terms of the rest of the above indicators （P<0.05， P<0.01）. Patients in neither groups complained of uncomfortable symptoms. ConclusionZhinao capsules have significant clinical efficacy in treating patients with mild and moderate sleep disorders in Alzheimer's disease patients with the syndrome of spleen-kidney deficiency and combined phlegm and stasis. Zhinao capsules can significantly reduce PSQI， ESS， and ADL scores， increase the MMSE score and the expression of BDNF and GABA， effectively optimize the structure of sleep， improve the intelligence， and enhance the ability of daily life， without causing uncomfortable symptoms. The mechanism may be related to the increase in the expression of sleep/wake-related neurotransmitters.

ObjectiveTo observe the clinical efficacy and safety of Zhinao capsules in the treatment of mild and moderate sleep disorders in Alzheimer's disease patients with the syndrome of spleen-kidney deficiency and combined phlegm and stasis. MethodsA randomized， double-blind， placebo， controlled clinical trial design was adopted， and 60 Alzheimer's disease patients aged 50-85 years with mild and moderate sleep disorders due to spleen-kidney deficiency and combined phlegm and stasis were included. According to the randomized numble table method， the patients were assigned into a treatment group and a control group （30 in each group， with 29 patients in the treatment group and 28 patients in the control group finally completing the trial）. The treatment group was treated with donepezil hydrochloride tablets combined with Zhinao capsules， and the control group was treated with donepezil hydrochloride tablets combined with the simulant of Zhinao capsules for 12 weeks. The Pittsburgh sleep quality index （PSQI）， smart bracelet sleep monitoring， epworth sleeping scale （ESS）， Mini-Mental State Examination （MMSE）， Activities of Daily Living （ADL）， and traditional Chinese medicine symptoms were scored. Furthermore， the brain-derived neurotrophic factor （BDNF） and gamma-aminobutyric acid （GABA） levels were measured. ResultsAfter 12 weeks of treatment， the total response rate of clinical efficacy assessment in the treatment group reached 89.7%， which was significantly higher than that （21.4%） in the control group （Z=-5.14， P<0.01）. After treatment， the TCM symptom scores declined in both groups （P<0.01）， and the scores in the treatment group were lower than those in the control group （P<0.01）. After treatment， the treatment group showed decreased total score and seven component scores of the PSQI scale （P<0.05， P<0.01）， and the scores in the treatment group were lower than those in the control group （P<0.05， P<0.01）. After treatment， the treatment group showed increases in the duration of deep sleep， light sleep， rapid eye movement sleep， and total sleep at night and decreases in the time to fall asleep， the number of awakenings from sleep at night， and the time for sporadic daytime naps （P<0.01）. The control group did not show significant changes in the duration of light sleep， time to fall asleep， and number of awakenings from sleep at night. Moreover， the treatment group outperformed the control group in terms of the indicators above （P<0.05， P<0.01）. After treatment， the treatment group showed decreased scores of ESS， ADL and its subscales physical self-maintenance scale （PSMS）， and Instrumental Activities of daily living scale （IADLs） and increased score of MMSE and levels of BDNF and GABA （P<0.05， P<0.01）. There was no significant difference in PSMS between the two groups， and the treatment group was significantly better than the control group in terms of the rest of the above indicators （P<0.05， P<0.01）. Patients in neither groups complained of uncomfortable symptoms. ConclusionZhinao capsules have significant clinical efficacy in treating patients with mild and moderate sleep disorders in Alzheimer's disease patients with the syndrome of spleen-kidney deficiency and combined phlegm and stasis. Zhinao capsules can significantly reduce PSQI， ESS， and ADL scores， increase the MMSE score and the expression of BDNF and GABA， effectively optimize the structure of sleep， improve the intelligence， and enhance the ability of daily life， without causing uncomfortable symptoms. The mechanism may be related to the increase in the expression of sleep/wake-related neurotransmitters.

ObjectiveTo investigate the effects of Dihuang Yinzi on the cognitive function in the mouse model of learning and memory impairments induced by scopolamine （SCOP） and explore the treatment mechanism. MethodsA mouse model of learning and memory impairment was induced by intraperitoneal injection of SCOP. Sixty male C57BL/6J mice were randomized into six groups： control （0.9% NaCl， n=10）， model （SCOP 1 mg·kg^-1·d^-1， n=10）， low-， medium-， and high-dose Dihuang Yinzi （SCOP 1 mg·kg^-1·d^-1 + Dihuang Yinzi 5.5， 11.0， and 22.0 g·kg^-1·d^-1， n=10）， and donepezil （SCOP 1 mg·kg^-1·d^-1 + donepezil 0.84 mg·kg^-1·d^-1， n=10）. Mice were administrated with corresponding drugs for 6 weeks. Modeling started in the 4th week， and mice in other groups except the control group were injected with SCOP intraperitoneally 40 min after daily gavage. Behavioral testing began in the 5th week， 30 min after modeling each day. The Morris water maze and novel object recognition tests were carried out to evaluate the spatial learning and memory function of mice. Nissl staining was employed to observe the survival of neurons and Nissl bodies in the hippocampal CA1 region. Western blot was employed to determine the protein levels of silent information regulator 2 （SIRT2）， α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid （AMPA） receptor 1 （GluA1）， protein kinase A （PKA）， cAMP response element-binding protein （CREB）， phosphorylated-CREB （p-CREB）， postsynaptic density protein 95 （PSD95）， growth-associated protein-43 （GAP-43）， and synaptophysin （SYN） in the hippocampus. Immunofluorescence was used to detect the expression of doublecortin （DCX） in the hippocampal dentate gyrus （DG） region. ResultsCompared with the control group， the model group showed impaired learning and memory （P<0.01）， obvious neuronal damage in the hippocampal CA1 region， a reduction in neuron survival （P<0.01）， a decrease in DCX expression in the hippocampal DG region （P<0.01）， down-regulated proteins levels of GluA1， PKA， p-CREB/CREB， PSD95， SYN， and GAP-43 in the hippocampal tissue （P<0.05， P<0.01）， and an up-regulated protein level of SIRT2 （P<0.01）. Compared with the model group， the medium- and high-dose Dihuang Yinzi groups and the donepezil group showed improvements in learning and memory （P<0.05， P<0.01）， while the low-， medium-， and high-dose Dihuang Yinzi groups and the donepezil group had increased neuron survival （P<0.05， P<0.01）. The medium-dose Dihuang Yinzi group and the donepezil group showed increased DCX expression （P<0.05， P<0.01）. The medium- and high-dose Dihuang Yinzi groups and the donepezil group showed up-regulation in the protein levels of GluA1， PKA， p-CREB/CREB， PSD95， SYN， and GAP-43 （P<0.05， P<0.01） and down-regulation in the protein level of SIRT2 （P<0.01）. ConclusionDihuang Yinzi can improve the cognitive function in the mouse model of learning and memory impairments induced by SCOP by inhibiting the upregulation of SIRT2， activating the PKA/CREB signaling pathway， improving synaptic plasticity， and reducing hippocampal neuronal damage.

Mild cognitive impairment due to Alzheimer′s disease is an inevitable pathological stage in the early development of Alzheimer′s disease, which can be classified as "microlumps in the brain collaterals" in traditional Chinese medicine. Based on the theory of latent toxin blocking collaterals, this article discusses the etiology and pathogenesis, clinical sequelae, and traditional Chinese medicine intervention strategies for mild cognitive impairment due to Alzheimer′s disease. The onset of mild cognitive impairment due to Alzheimer′s disease is very similar to the latent pathogen theory, which states that "the latent pathogen is latent and then develops, the poison is deep and difficult to cure, and the development can be recognized but the latent pathogen cannot be detected." Combining clinical experience, our team believes that the basic nature of the disease is a slight deficiency and a slight excess of symptoms. A slight deficiency of the five zang viscera and six fu viscera as root and a latent toxin colling collaterals of qi, fire, phlegm, and blood stasis as manifestaion. These usually start from the qi depression and develop into phlegm coagulation and blood stasis, then end up in latent toxin and gradually become the healthy qi deficiency. Therefore, the deficiency of vital qi and incubation of evil, latent toxin blocking collaterals the pathogenesis of early intervention of this disease should be carried out, upholding the idea that "the upper workman treats the disease before it is diagnosed." The principle of strengthening vital qi to eliminate pathogenic factors, slowing down and promoting pathogenic factors elimination, establishing the method of supporting correctness and wisdom, simultaneously detoxifying and clearing the blood stasis, pattern differentiation as the main and the disease differentiation as the first, combining the disease and pattern, and adjusting the macroscopic and microscopic, focusing simultaneously on eliminating and replenishing, dispel phlegm and remove blood stasis, achieve a strong vital qi and the elimination of evil, and enhance intelligence, delay or even block the progression of mild cognitive impairment due to Alzheimer′s disease, improve patients′ quality of life, and provide a theoretical basis for the early clinical prevention and treatment of Alzheimer′s disease.

Objective:To identify the main components of Huanglian Jiedu Decoction(HLJDD)using ultra-high-performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-Q-TOF-MS),and to explore the potential mechanism of action of HLJDD in the treatment of gouty arthritis(GA)using network pharmacology and molecular docking methods.Methods:We identi-fied the chemical components of HLJDD by combining UPLC-Q-TOF-MS data acquired in both positive and negative ion modes with reference standards,relevant literature,and database searches.We analyzed the potential therapeutic mechanism of HLJDD for GA by using network pharmacology to determine the intersection targets between the active ingredients of HLJDD and GA for further enrich-ment analysis and visual network mapping.The binding affinity of the active ingredients with the intersection targets was validated through molecular docking.Results:A total of 47 components were identified by UPLC-Q-TOF-MS;54 key components of HLJDD for GA treatment and 37 intersection targets were determined by net-work pharmacology;and the top 10 key targets by Degree value were obtained by protein-protein interaction analysis.The Gene On-tology functional enrichment analysis revealed 20 biological pro-cesses,7 cellular components,and 8 molecular functions.The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated 96 GA-related intervention pathways,in which inflammatory signaling pathways such as interleukin-17(IL-17)and tu-mor necrosis factor(TNF)were involved.Molecular docking verified that the key components of HLJDD had high binding affinity with the core targets.Conclusion:The identified key components in HLJDD,such as phellodendrine,coptisine,wogonin,and β-sitosterol,may alleviate GA by regulating multiple core targets in the IL-17 and TNF pathways,such as PTSG2,which provides a theoretical ba-sis for future investigation into the mechanism of action of HLJDD.

Chlorobenzene contaminants (CBs) pose a threat to the eco-environment, and functional strains hold considerable potential for the remediation of CB-contaminated sites. To deeply explore the application potential of functional bacteria in the in-situ bioremediation of CBs, this study focused on the biodegradation characteristics and degradation kinetics of CB and 1, 2-dichlorobenzene (1, 2-DCB) in soil by the isolated strain Serratia marcescens TF-1. Additionally, an in-situ remediation trial was conducted with this strain at a chemical industrial site. Batch serum bottle experiments showed that the degradation rate of CB at the concentrations ranging from 20 to 200 mg/L by TF-1 was 0.22-0.66 mol/(g_cell·h), following the Haldane model, with the optimal concentration at 23.12 mg/L. The results from simulated soil degradation experiments indicated that the combined use of TF-1 and sodium succinate (SS) significantly enhanced the degradation of CBs, with the maximum degradation rate of CB reaching 0.104 d^-1 and a half-life of 6.66 d. For 1, 2-DCB, the maximum degradation rate constant was 0.068 7 d^-1, with a half-life of 10.087 d. The in-situ remediation results at the chemically contaminated site demonstrated that the introduction of bacterial inoculant and SS significantly improved the removal of CBs, achieving the removal rates of 84.2%-100% after 10 d. CB, 1, 4-dichlorobenzene (1, 4-DCB), and benzo[a]pyrene were completely removed. Microbial diversity analysis revealed that the in-situ remediation facilitated the colonization of TF-1 and the enrichment of indigenous nitrogen-fixing Azoarcus, which may have played a key role in the degradation process. This study provides a theoretical basis and practical experience for the in situ bioremediation of CBs-contaminated sites.
Chlorobenzenes/isolation & purification* ; Biodegradation, Environmental ; Soil Pollutants/isolation & purification* ; Serratia marcescens/metabolism* ; Industrial Waste ; Soil Microbiology

Objective:To explore the influence of Stat1 on Foxp3 expression and production of Treg.Methods:C57BL/6 mice were used and separated into normal control group and Stat1 specific inhibitor Fludarabine(Flud)treatment group.Ratio of CD4+Foxp3+Treg and expression of Foxp3 in spleen,lymph nodes and peripheral blood of mice in each group were detected by flow cy-tometry.Human Stat1 overexpression plasmid was constructed and transfected into human breast cancer MCF-7 cells,and expression changes of Foxp3 was detected by RT-qPCR and Western blot.Results:Compared with mice in normal control group,proportion of Treg and expression of Foxp3 in lymph nodes and peripheral blood of mice in Flud treatment group were increased,while Stat1 overex-pression resulted in decreased Foxp3 mRNA and protein expression in MCF-7 cells.Conclusion:Stat1 inhibits expression of Foxp3 and production of Tregs.

To investigate the in vitro inhibitory mechanism of Nymphaea candida total flavonoids(NCTF)against Staphylococcus aureus(S.aureus)and its safety in mice,this study first deter-mined the antibacterial effect of NCTF on the clinically isolated strain S.aureus-C1.Subsequently,the inhibitory mechanism of NCTF on S.aureus-C1 was explored by measuring its effects on bac-terial growth curves,microstructure,intracellular AKP and LDH levels,and biofilm formation.Safety evaluation included determination of LD50 and MDT in mice,as well as analysis of serum biochemical parameters,organ indices,and histopathological observations.Results showed that NCTF effectively inhibited S.aureus-C1 proliferation,with an inhibition zone diameter of(18.98±0.67)mm and a MIC of 6.25 g/L.A concentration of 2×MIC nearly completely suppressed bacte-rial growth.Scanning electron microscopy revealed structural damage to bacterial cells,including collapse and shrinkage.AKP and LDH assays indicated significantly increased AKP activity(P＜0.05)and decreased intracellular LDH activity(P＜0.05)in the supernatant of drug-treated groups,demonstrating NCTF-induced disruption of cell walls and membranes leading to leakage of AKP and LDH.Crystal violet staining of biofilms showed significant inhibition rates of(43.77±9.16)％and(61.71±9.82)％at 2 × MIC and 4 × MIC concentrations,respectively(P＜0.05).Safe-ty assessments indicated low toxicity of NCTF in mice,with transient effects that returned to nor-mal levels within a short period.These findings demonstrate that NCTF exhibits potent antibacte-rial activity against S.aureus-C1 by damaging bacterial cell structures,increasing cell wall/mem-brane permeability,reducing biofilm formation,and displaying low toxicity.This study provides scientific evidence for clinical drug screening against bovine mastitis and the development of Nym-phaea candida resources.

To investigate the in vitro inhibitory mechanism of Nymphaea candida total flavonoids(NCTF)against Staphylococcus aureus(S.aureus)and its safety in mice,this study first deter-mined the antibacterial effect of NCTF on the clinically isolated strain S.aureus-C1.Subsequently,the inhibitory mechanism of NCTF on S.aureus-C1 was explored by measuring its effects on bac-terial growth curves,microstructure,intracellular AKP and LDH levels,and biofilm formation.Safety evaluation included determination of LD50 and MDT in mice,as well as analysis of serum biochemical parameters,organ indices,and histopathological observations.Results showed that NCTF effectively inhibited S.aureus-C1 proliferation,with an inhibition zone diameter of(18.98±0.67)mm and a MIC of 6.25 g/L.A concentration of 2×MIC nearly completely suppressed bacte-rial growth.Scanning electron microscopy revealed structural damage to bacterial cells,including collapse and shrinkage.AKP and LDH assays indicated significantly increased AKP activity(P＜0.05)and decreased intracellular LDH activity(P＜0.05)in the supernatant of drug-treated groups,demonstrating NCTF-induced disruption of cell walls and membranes leading to leakage of AKP and LDH.Crystal violet staining of biofilms showed significant inhibition rates of(43.77±9.16)％and(61.71±9.82)％at 2 × MIC and 4 × MIC concentrations,respectively(P＜0.05).Safe-ty assessments indicated low toxicity of NCTF in mice,with transient effects that returned to nor-mal levels within a short period.These findings demonstrate that NCTF exhibits potent antibacte-rial activity against S.aureus-C1 by damaging bacterial cell structures,increasing cell wall/mem-brane permeability,reducing biofilm formation,and displaying low toxicity.This study provides scientific evidence for clinical drug screening against bovine mastitis and the development of Nym-phaea candida resources.

Objective:To explore the influence of Stat1 on Foxp3 expression and production of Treg.Methods:C57BL/6 mice were used and separated into normal control group and Stat1 specific inhibitor Fludarabine(Flud)treatment group.Ratio of CD4+Foxp3+Treg and expression of Foxp3 in spleen,lymph nodes and peripheral blood of mice in each group were detected by flow cy-tometry.Human Stat1 overexpression plasmid was constructed and transfected into human breast cancer MCF-7 cells,and expression changes of Foxp3 was detected by RT-qPCR and Western blot.Results:Compared with mice in normal control group,proportion of Treg and expression of Foxp3 in lymph nodes and peripheral blood of mice in Flud treatment group were increased,while Stat1 overex-pression resulted in decreased Foxp3 mRNA and protein expression in MCF-7 cells.Conclusion:Stat1 inhibits expression of Foxp3 and production of Tregs.