Objective: To investigate the effect of auricular therapy on sleep improvement and the GABAergic system pathway in a rat model of insomnia and to explore its possible mechanism. Methods: According to the random number table, 60 male SD rats were randomly divided into blank control, model, auricular point sticking, auricular bloodletting, and auricular bloodletting combined with sticking groups, with 12 rats per group. Insomnia was induced by intraperitoneal injection of p-chlorophenylalanine. After establishing the insomnia model, 36 rats were treated once a day with auricular point sticking or bloodletting for 5 consecutive days. After the intervention, the general condition and body weight of rats were observed; the righting reflex test was used to detect the sleep latency and duration; HE staining was used to observe the morphology of hypothalamic neuron cells; and an enzyme-linked immunosorbent assay was used to detect the GABA and glutamate content in rat serum. Immunohistochemistry(IHC) and real-time fluorescence quantitative PCR were used to detect GABA ARα1 and GABA ARγ2 protein and mRNA expression in the hypothalamus of rats, and Western blotting(WB) was used to detect GABA ARα1, GABA ARγ2, GAD65/67, GAT-1, and GABA-T protein expression in the hypothalamus of rats. Results: Compared with the blank control group, the model group had a lower body weight, a significantly shorter sleep duration (P<0.05), severe damage to the morphological structure of hypothalamic neurons with disordered cell arrangement, larger intercellular gaps, enlarged cell bodies, and a vacuolated appearance. All the intervention groups had significantly higher body weight and longer sleep duration than the model group (P<0.05). Compared with the other intervention groups, the auricular point sticking group had a longer sleep duration (P<0.05), and the hypothalamic neuron cells in all intervention groups improved, with the auricular point sticking group showing more apparent improvement. The model group had a lower GABA and higher glutamate contents, and GABA ARα1, GABA ARγ2, and GAD65/67 protein expression in the hypothalamus were lower than in the blank control group. In contrast, GAT-1 and GABA-T protein expression was higher, and GABA ARα1 and GABA ARγ2 mRNA expression was lower (P<0.05). The serum GABA content in the auricular point sticking and auricular bloodletting groups was higher, and the serum glutamate content in the auricular point sticking and auricular bloodletting combined sticking groups was lower than in the model group. GABA ARα1 mRNA expression in the hypothalamus of each intervention group was significantly increased, and GABA ARγ2 mRNA expression in the hypothalamus of the auricular point sticking and auricular bloodletting combined sticking groups increased. GABA ARα1(IHC, WB), GABA ARγ2(WB), and GAD65/67 protein expression in the hypothalamus of the auricular point sticking group increased, whereas GAT-1 and GABA-T protein expression decreased. GABA ARα1 and GABA ARγ2 protein expression(IHC, WB) in the hypothalamus of the auricular bloodletting group increased, whereas GABA-T protein expression decreased. GABA ARγ1(IHC) and GABA ARγ2(WB) protein expression in the hypothalamus of the auricular bloodletting combined sticking group increased, whereas GAT-1 and GABA-T protein expression decreased (P<0.05). Compared with in the inventation groups, the serum GABA content in the auricular point sticking group increased, the serum glutamate content decreased, GABA ARα1 and GABA ARγ2 mRNA expression in the hypothalamus increased, and GABA ARα1(IHC), GAD65/67 protein expression increased. In contrast, GABA-T protein expression decreased (P<0.05), and GABA ARγ2 protein expression(IHC) in the hypothalamus of the auricular bloodletting group increased (P<0.05). Conclusion Auricular therapy, particularly auricular point sticking, may have modulated the GABAergic system pathway by upregulating hypothalamic GABA ARα1, GABA ARγ2, and GAD65/67 protein expression while downregulating GAT-1 and GABA-T protein expression to alleviate symptoms in an insomnia rat model.

HDAC7, a member of class IIa HDACs, plays a pivotal regulatory role in tumor, immune, fibrosis, and angiogenesis, rendering it a potential therapeutic target. Nevertheless, due to the high similarity in the enzyme active sites of class IIa HDACs, inhibitors encounter challenges in discerning differences among them. Furthermore, the substitution of key residue in the active pocket of class IIa HDACs renders them pseudo-enzymes, leading to a limited impact of enzymatic inhibitors on their function. In this study, proteolysis targeting chimera (PROTAC) technology was employed to develop HDAC7 drugs. We developed an exceedingly selective HDAC7 PROTAC degrader B14 which showcased superior inhibitory effects on cell proliferation compared to TMP269 in various diffuse large B cell lymphoma (DLBCL) and acute myeloid leukemia (AML) cells. Subsequent investigations unveiled that B14 disrupts BCL6 forming a transcriptional inhibition complex by degrading HDAC7, thereby exerting proliferative inhibition in DLBCL. Our study broadened the understanding of the non-enzymatic functions of HDAC7 and underscored the importance of HDAC7 in the treatment of hematologic malignancies, particularly in DLBCL and AML.

Objective: To assess the dynamic changes of microcirculation at acupoints in patients with primary dysmenorrhea and cold congelation and blood stasis syndrome using laser speckle blood flow imaging. Methods: Patients with primary dysmenorrhea and cold coagulation and blood stasis syndrome (primary dysmenorrhea group, n=53) and healthy female college students(control group, n=57) who met the inclusion and exclusion criteria from October 2020 to July 2022 were enrolled at Hebei University of Chinese Medicine. On the premenstrual and first day of menstruation, a laser speckle blood flow imaging system was used to measure the microcirculation blood flow perfusion on the surface of acupoints related to the conception, thoroughfare, and governor vessels, and stomach, spleen, and bladder meridians in the abdomen and lumbosacral regions. The dynamic changes in microcirculation were calculated based on the difference in average blood flow perfusion at each acupoint before and after menstruation. Receiver operating curve (ROC) analysis was used to analyze the diagnostic efficacy of dynamic changes in microcirculation on the surface of each acupoint. The microcirculation sensitization rate of acupoints was calculated. Results: Compared with the control group, the dynamic changes in microcirculation at the following acupoints in the primary dysmenorrhea group were increased (P<0.05): conception vessel (Yinjiao[CV7], Qihai[CV6], Shimen[CV5], Guanyuan[CV4]); left thoroughfare vessel (left Huangshu[KI16], left Zhongzhu[KI15], left Siman[KI14], left Qixue[KI13], left Dahe[KI12], left Henggu[KI11]); left stomach meridian (left Tianshu[ST25], left Wailing[ST26], left Qichong[ST30]); left spleen meridian (left Daheng[SP15], left Fujie[SP14]); right thoroughfare vessel (right Huangshu[KI16], right Zhongzhu[KI15], right Siman[KI14], right Qixue[KI13], right Dahe[KI12], right Henggu[KI11]); right stomach meridian (right Wailing[ST26], right Daju[ST27], right Shuidao[ST28], right Guilai[ST29], right Qichong[ST30]); and right spleen meridian (right Fujie[SP14]). The area under the ROC curve of conception vessel (Yinjiao[CV7], Qihai[CV6], Shimen[CV5], Guanyuan[CV4]), thoroughfare vessel (right Siman[KI14], left Huangshu[KI16], right Qixue[KI13], right Zhongzhu[KI15], right Dahe[KI12], left Zhongzhu[KI15], left Siman[KI14], right Huangshu[KI16], left Qixue[KI13], right Henggu[KI11], left Henggu[KI11], left Dahe[KI12]); stomach meridian (left Tianshu[ST25], right Guilai[ST29], left Wailing[ST26], right Shuidao[ST28], right Daju[ST27], right Wailing[ST26], right Qichong[ST30], left Qichong[ST30]), and spleen meridian (left Daheng[SP15], left Fujie[SP14], right Fujie[SP14]) was 0.610-0.682 (P<0.05). Compared with the control group, the sensitization rate of some acupoints in the primary dysmenorrhea group increased (P<0.05). Conclusion With the onset of menstruation, the blood flow perfusion of some acupoints in the abdomen (thoroughfare, and conception vessels, and stomach and spleen meridians) of patients with primary dysmenorrhea and cold blood coagulation and blood stasis syndrome increased, and the status of acupoints changed from a resting state to an active state. These acupoints are sensitive in patients with primary dysmenorrhea and cold blood coagulation and blood stasis syndrome and have a certain diagnostic efficacy, providing a basis for further analyzing the efficacy and mechanism of acupuncture and moxibustion to treat primary dysmenorrhea with cold blood coagulation and blood stasis syndrome.

Objective:To investigate the effect of bone morphogenetic protein-9(BMP9)overexpression on inflammatory response and apoptosis of alveolar epithelial cells induced by lipopolysaccharide(LPS).Methods:A549 cells were stimulated with 0.1 μg/ml LPS,expressions and changes of BMP9 protein at different time points(LPS stimulation for 0 h,6 h,12 h,24 h)were detected by Western blot.Expression of BMP9 in A549 cells was up-regulated by transfection of BMP9 plasmid,and the transfection efficiency was verified by Western blot and qPCR.After 12 h of LPS stimulation,levels of inflammatory factors TNF-α,IL-6,IL-1β in cell supernatant were detected by ELISA,expressions of anti-apoptotic protein(Bcl-2)and pro-apoptotic protein(Bax)were detected by Western blot,and apoptosis of cells was detected by TUNEL staining.Results:With the extension of LPS stimulation time,expression of BMP9 was down-regulated.Overexpression of BMP9 successfully up-regulated expression of BMP9 in A549 cells.LPS stimulation promoted secretions of TNF-α,IL-6 and IL-1β from A549 cells,increased apoptosis,promoted Bcl-2 expression while inhibited Bax expression.Overexpression of BMP9 inhibited TNF-α,IL-6 and IL-1β releasing,decreased apoptosis,inhibited Bcl-2 expression,while promoted Bax expression.Conclusion:In LPS-stimulated A549 cells,BMP9 expression is gradually decreased at a time-depen-dent manner.Overexpression of BMP9 can inhibit LPS-induced inflammatory response and apoptosis in A549 cells.

Objective:To investigate the effect of bone morphogenetic protein-9(BMP9)overexpression on inflammatory response and apoptosis of alveolar epithelial cells induced by lipopolysaccharide(LPS).Methods:A549 cells were stimulated with 0.1 μg/ml LPS,expressions and changes of BMP9 protein at different time points(LPS stimulation for 0 h,6 h,12 h,24 h)were detected by Western blot.Expression of BMP9 in A549 cells was up-regulated by transfection of BMP9 plasmid,and the transfection efficiency was verified by Western blot and qPCR.After 12 h of LPS stimulation,levels of inflammatory factors TNF-α,IL-6,IL-1β in cell supernatant were detected by ELISA,expressions of anti-apoptotic protein(Bcl-2)and pro-apoptotic protein(Bax)were detected by Western blot,and apoptosis of cells was detected by TUNEL staining.Results:With the extension of LPS stimulation time,expression of BMP9 was down-regulated.Overexpression of BMP9 successfully up-regulated expression of BMP9 in A549 cells.LPS stimulation promoted secretions of TNF-α,IL-6 and IL-1β from A549 cells,increased apoptosis,promoted Bcl-2 expression while inhibited Bax expression.Overexpression of BMP9 inhibited TNF-α,IL-6 and IL-1β releasing,decreased apoptosis,inhibited Bcl-2 expression,while promoted Bax expression.Conclusion:In LPS-stimulated A549 cells,BMP9 expression is gradually decreased at a time-depen-dent manner.Overexpression of BMP9 can inhibit LPS-induced inflammatory response and apoptosis in A549 cells.

Objective To construct a short hairpin shRNA recombinant lentiviral vector targeting mouse VASP gene to interfere with the expression of VASP in mouse alveolar macrophage line(MH-S)cells.Methods shRNA targeting mouse VASP gene were de-signed,inserted into the shuttle plasmid GV644-EGFP vector,and identified by polymerization chain reaction(PCR)and sequencing.The recombinant plasmids GV644-VASP-shRNA-EGFP,auxiliary packaging pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells for packaging of recombinant lentivirus lenti-GV644-VASP-shRNA-EGFP,and titers were determined by fluorescent la-beling.MH-S cells were transfected with lenti-VASP-shRNA-EGFP at different multiple of infection(MOI)values,and the optimal MOI value was obtained according to the infection efficiency,and the expression of the target gene was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot.Results The shuttle plasmid GV644-VASP-shRNA-EGFP was successfully constructed by PCR and sequencing identification,and the recombinant lenti-VASP-shRNA-EGFP was further pack-aged with a viral titer of 2 × 109/(TU·ml),and the optimal MOI value of MH-S cells was 50,which significantly inhibited the expres-sion of VASP after infection with MH-S cells(P＜0.05).Conclusion In this study,the shRNA recombinant lentiviral vector targeting mouse VASP gene was successfully constructed,which significantly inhibited the expression of VASP in mouse MH-S cells,which laid a foundation for further exploring the role of VASP gene in macrophage phenotype regulation.

Objective:To evaluate the clinical efficacy of acupuncture in the treatment of anxiety state in patients with primary insomnia(PI). Methods:Randomized controlled trials of acupuncture treatment for PI patients with an anxiety state in Web of Science,Cochrane Library,PubMed,Excerpta Medica Database(EMBASE),China National Knowledge Infrastructure(CNKI),Wanfang Data Knowledge Service Platform(Wanfang),and Chongqing VIP Database(VIP)were retrieved by computer.The retrieval time was from each database's inception to December 30,2022.Data extraction and evaluation were performed for the included studies.The Cochrane risk of bias assessment tool was used to assess the risk of bias in each article.Meta-analysis of valid data was performed using the RevMan 5.4 software.If the outcome indicator was a categorical variable,relative risk(RR)was used as the effect size.If it was a continuous variable,mean difference(MD)was used to calculate the effect size.Each effect size was expressed as a 95%confidence interval(CI).P<0.05 was considered to indicate a statistically significant difference. Results:A total of 18 studies were included,comprising a total of 1198 patients.The findings of the meta-analysis showed that electroacupuncture had a significant advantage in improving the Hamilton anxiety scale(HAMA)score than benzodiazepines[MD=-1.61,95%CI(-2.17,-1.06),P<0.001].Acupuncture was superior to sham acupuncture[MD=-14.90,95%CI(-20.39,-9.41),P<0.001]and benzodiazepines[MD=-3.39,95%CI(-4.67,-2.12),P<0.001]in reducing the self-rating anxiety scale(SAS)score.Acupuncture was superior to sham acupuncture in reducing the insomnia severity index(ISI)score[MD=-5.61,95%CI(-6.63,-4.89),P<0.001].Acupuncture was superior to benzodiazepines[MD=0.84,95%CI(-1.42,-0.25),P=0.005]and sham acupuncture[MD=-8.39,95%CI(-8.39,-7.86),P<0.001]in improving the Pittsburgh sleep quality index(PSQI)score.Acupuncture had a better effective rate than benzodiazepines[RR=1.16,95%CI(1.08,1.25),P<0.001]and sham acupuncture[RR=8.94,95%CI(4.63,17.25),P<0.001]in treating PI. Conclusion:Acupuncture or electroacupuncture has certain therapeutic advantages over benzodiazepines and sham acupuncture in the treatment of anxiety in PI patients.However,more high-quality randomized controlled trials are needed for further verification.

Objective To construct a short hairpin shRNA recombinant lentiviral vector targeting mouse VASP gene to interfere with the expression of VASP in mouse alveolar macrophage line(MH-S)cells.Methods shRNA targeting mouse VASP gene were de-signed,inserted into the shuttle plasmid GV644-EGFP vector,and identified by polymerization chain reaction(PCR)and sequencing.The recombinant plasmids GV644-VASP-shRNA-EGFP,auxiliary packaging pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells for packaging of recombinant lentivirus lenti-GV644-VASP-shRNA-EGFP,and titers were determined by fluorescent la-beling.MH-S cells were transfected with lenti-VASP-shRNA-EGFP at different multiple of infection(MOI)values,and the optimal MOI value was obtained according to the infection efficiency,and the expression of the target gene was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot.Results The shuttle plasmid GV644-VASP-shRNA-EGFP was successfully constructed by PCR and sequencing identification,and the recombinant lenti-VASP-shRNA-EGFP was further pack-aged with a viral titer of 2 × 109/(TU·ml),and the optimal MOI value of MH-S cells was 50,which significantly inhibited the expres-sion of VASP after infection with MH-S cells(P＜0.05).Conclusion In this study,the shRNA recombinant lentiviral vector targeting mouse VASP gene was successfully constructed,which significantly inhibited the expression of VASP in mouse MH-S cells,which laid a foundation for further exploring the role of VASP gene in macrophage phenotype regulation.

Objective To explore the impact of glucocorticoid on coagulation through administrating on rats with smoke inhalation.Methods Totally 150 male S-D rats were randomly (random number) divided into 5 groups:control group (ambient air inhalation),smoke group (smoke inhalation for 30 min),smoke+high dosage methyl prednisolone group(MP 40 mg/kg,intraperitoneal injection,s+HMP group),smoke+medium dosage MP (4 mg/kg) group (s+MMP group),smoke+low dosage MP (0.4 mg/kg) group (s+LMP group) (all n=30).Survival rates were calculated 24 h after smoke inhalation.Lung tissues were collected for histopathology and wet to dry (W/D) ratio.Arterial blood was collected for blood gas test.Coagulation factors in lung and plasma were tested.Results Survival rates of three MP groups were markedly improved compared with the smoke group (all P＜0.05),and was significantly higher in the medium dosage group(85.17％) than those in the low and high dosage groups (65.73％ and 60.07％,all P＜0.05).The W/D ratio and blood gas test were markedly improved in the high and medium groups (all P＜0.05).Tissue factor (TF) and thrombin-antithrombin complex (TAT-c) in bronchoalveolar lavage fluid (BALF) increased dramatically after SI (P＜0.01,P=0.005) with a remarkable drop of factor Ⅱ (F Ⅱ) (P=0.007),all of which were attenuated by MP with dosage dependence.The mRNA expression of TF increased dramatically after SI and recovered significantly with MP administration,while the expression of thrombomodulin (TM) recovered in the opposite direction with MP,all of which were in a dosage dependent manner.TF,fibrinogen (FIB),TAT-c increased significantly in plasma after smoke inhalation (P＜0.01,P=0.027,P=0.005).F Ⅷ ％ increased with MP administration and TF was raised by high dosage MP compared with the smoke group.FIB and TAT-c were decreased in all MP groups,which were significant higher in the high and middle dosage groups.The change of TM and endothelial cell protein C receptor (EPCR) in circulation were similar with FIB or TAT-c with or without MP.Protein C (PC％) and antithrombin (AT Ⅲ ％) dropped dramatically after SI,high and middle dosages of MP could restore the activity significantly,while low dosage would restore AT Ⅲ ％ but not PC％.Conclusions Glucocorticoid can significantly improve local and systemical coagulation disorder caused by smoke inhalation,and high-and medium-dosage hormones are effective.The regulation of hormones on the coagulation system is an important mechanism in the treatment of smoke inhalation induced lung injury.

Objective To investigate the therapeutic effect of different doses of methylprednisolone (MP) in smoke inhalation-induced acute lung injury (SI-ALI).Methods Adult male Sprague-Dawley (SD) rats were divided into control group (group A,n = 6), smoke inhalation group (group B, smoke inhalation 30 minutes,n = 30) and smoke+MP 40, 4, 0.4 mg/kg intervention group (groups C, D, E; intraperitoneal injection of MP at 1 hour before smoke inhalation, n = 30) according to random number table method. The survival status of rats in each group was observed at 24 hours, and murine smoke inhalation induced trauma score (MSITS) according to the symptoms and signs of rats at 3 hours after smoke inhalation were scored. The blood of abdominal aorta of rats was collected. Then the rats were sacrificed to harvest bronchoalveolar lavage fluid (BALF) and lung tissue. The levels of interleukin (IL-6, IL-17a) in plasma and BALF were detected by enzyme linked immunosorbent assay (ELISA); the total number of white blood cells and the proportion of leukocytes or macrophages in BALF were calculated; the histopathological changes of lung were observed and the lung injury score was given; the expression of myeloperoxidase (MPO) and high mobility group protein B1 (HMGB1) in lung tissue were detected by Western Blot.Results The 24-hour survival rate of group B rats was 33.67％. The survivalrate of groups C, D and E (65.73％, 85.17％, 60.07％) were significantly higher than that of group B (allP < 0.05), and the survival rate of group D was significantly higher than that of groups C and E. Diffuse inflammatory cell infiltration, intra-alveolar hemorrhage and a large amount of edema fluid were seen in the lung tissue of group B; and the lung injury score was significantly higher than that of group A. Compared with group B, the lung injury in different doses of MP group were decreased to different degrees, while the lung injury scores in groups C and D were significantly decreased (3.31±1.37, 2.62±0.98 vs. 5.52±0.97, bothP < 0.01); correlation analysis showed that MSITS score was significantly and positively correlated with lung injury score (r = 0.862,P < 0.001). The levels of plasma inflammatory factors and BALF protein, inflammatory cells and inflammatory factors, and the expression of MPO, HMGB1 in group B were significantly higher than those in group A. Compared with group B, the levels of inflammatory factors in plasma, and protein content, inflammatory cells and inflammatory factors in BALF in different doses of MP group were decreased to different degrees, with significant differences in groups C and D [plasma: IL-17a (pg/L): 49.28±27.12, 36.57±16.52 vs. 191.79±88.21; IL-6 (ng/L): 206.47±109.96, 197.52±113.86 vs. 669.00±299.60; BALF: protein content (mg/L):892.0±164.5, 566.1±120.9 vs. 1838.0±145.8; white blood cell count (×109/L): 5.40±1.67, 2.81±1.20 vs. 9.02± 2.06; neutrophil ratio: 0.315±0.081, 0.273±0.080 vs. 0.590±0.096; IL-17a (ng/L): 22.63±8.62, 18.92±8.43 vs. 43.31±19.17; IL-6 (ng/L): 156.49±46.94, 123.66±64.91 vs. 253.43±80.03; allP< 0.01]; in addition, the expression of MPO and HMGB1 protein in lung tissues of MP groups with different doses were significantly decreased, the expression of MPO in group D was significantly lower than that in group E [MPO/β-actin (fold increase from group A):2.14±0.97 vs. 4.35±0.87,P < 0.01], the expression of HMGB1 in groups C and D were significantly lower than that in group E [HMGB1/β-actin (fold increase from group A): 1.77±0.73, 1.23±0.67 vs. 3.65±1.08, bothP < 0.05]. Conclusions MP can significantly improve the survival rate of SI-ALI rats and reduce the acute pulmonary and systemic inflammatory response. The MP effect of 4 mg/kg was better than 40 mg/kg and 0.4 mg/kg.