1.Primary exploration of stage I anastomosis and T-tube fistulation in laparoscopic local resection of duodenal tumors
Lijie LUO ; Tao WANG ; Xinrui YE ; Xianzhe WANG ; Zhuoxuan ZHANG ; Zijing ZHANG ; Yaohui PENG ; Yan CHEN ; Haiping ZENG ; Haipeng TANG ; Jiantao LIN ; Weiqiang ZOU ; Wei WANG
Chinese Journal of Gastrointestinal Surgery 2025;28(2):198-202
Objective:To discuss the feasibility and safety of stage I anastomosis and T-tube fistulation in laparoscopic local resection of duodenal tumors.Methods:A descriptive case series study was used to retrospectively analyze the clinical diagnosis and treatment data of 14 patients with duodenal tumors who successfully underwent laparoscopic local resection of duodenal tumors + phase I anastomosis + T-tube ostomy in the Guangdong Provincial Hospital of Chinese Medicine and the First Affiliated Hospital of Guangzhou University of Chinese Medicine from October 2021 to March 2024. The resection and reconstruction steps of laparoscopic local resection of duodenal tumor + phase I anastomosis + T-tube ostomy are as follows: (1) after the safe margin is clear, the duodenal tumor is completely removed in full thickness, and the specimen bag is taken out and sent to frozen section to determine the nature of the tumor and the negative margin; (2) Perforate the anterior duodenal wall below the tumor plane, place a 16# T tube, and fix it with laparoscopic purse string suture. The abdominal wall is led out through the duodenum, and the duodenal T tube fistulation is performed; (3) The duodenum was continuously sutured in a full-thickness transverse shape, and the seromuscular layer was strengthened to form a phase I anastomosis. The nutritional improvement of patients after operation was mainly observed, and the intraoperative situation and postoperative complications were recorded.Results:No conversion to laparotomy, postoperative emergency reoperation, intraoperative and postoperative complications occurred in 14 patients with duodenal tumors who completed laparoscopic local resection of duodenal tumors + phase I anastomosis + T-tube ostomy. The operation time was (225.43 ± 56.54) min, and the intraoperative blood loss was (72.14 ± 74.65) ml. The patient recovered well after operation, and no severe postoperative abdominal bleeding occurred. Postoperative gastrointestinal angiography showed that the anastomotic stoma was unobstructed, and there were no stenosis, anastomotic leakage and other related complications. There was no significant difference in serum albumin [(37.09 ± 3.53) g/L vs. (37.52 ± 4) g/L] and hemoglobin [(100.79 ± 31.93) g/L vs. (103.07 ± 19.6) g/L] between before and 1 week after operation ( P > 0.05). Conclusion:Laparoscopic local resection of duodenal tumor + phase I anastomosis + T-tube fistulation can be used as one of the safe and feasible improved methods for local resection of duodenal tumor to effectively reduce the occurrence of related complications.
2.Mechanism of Anmeidan in Improving Learning and Memory in Insomnia Model Rats by Mediating Immunoinflammation via cGAS/STING Signaling Pathway
Bo XU ; Zijing YE ; Ping WANG ; Jing CHENG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(10):27-35
ObjectiveTo investigate the mechanism by which Anmeidan improves learning and memory in insomnia rats by regulating the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway to influence immunoinflammation. MethodsSixty SD rats were randomly divided into a blank group, a model group, a suvorexant group (30 mg·kg-1), and Anmeidan low-, medium-, and high-dose groups (4.55, 9.09, and 18.18 g·kg-1), with 10 rats in each group. The insomnia rat model was induced by intraperitoneal injection of p-chlorophenylalanine (PCPA). Anmeidan decoction and normal saline were administered by gavage for 28 days at the corresponding doses. Morris water maze and new object recognition tests were used to assess learning and memory functions. Hematoxylin-eosin (HE) staining and Nissl staining were performed to observe hippocampal cell morphology. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum levels of interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α). Western blot and Real-time quantitative polymerase chain reaction(Real-time PCR) were used to detect the relative protein and mRNA expression levels of hippocampal cGAS and STING. ResultsCompared with the blank group, the 5-HT content in the model group was significantly reduced (P<0.01). The latency to the upper platform and total distance were significantly increased (P<0.05, P<0.01), while the residence time in the target quadrant and the number of platform crossings were significantly reduced (P<0.01), and the relative recognition index for new objects was significantly lower (P<0.01). The morphology and arrangement of hippocampal neurons were loose and disordered, with a decreased number of intracellular Nissl bodies. The relative expression levels of IL-1, IL-1β, IL-6, IL-8, IL-12, IL-18, TNF-α, cGAS, and STING pathway proteins and mRNA were significantly upregulated (P<0.01). Compared with the model group, the latency to the upper platform in the high-dose Anmeidan group was significantly shortened (P<0.05). In the medium- and high-dose Anmeidan groups and the suvorexant group, the residence time in the target quadrant and the number of platform crossings were significantly increased (P<0.01). The total distance traveled was significantly reduced (P<0.01), and the relative recognition index for new objects was significantly increased (P<0.01). The hippocampal neurons were more neatly arranged, and the number of intracellular Nissl bodies increased. The expression of IL-1, IL-1β, IL-6, IL-8, IL-12, IL-18, TNF-α, and cGAS proteins and mRNA in the medium- and high-dose Anmeidan groups was significantly downregulated (P<0.05, P<0.01). ConclusionAnmeidan improves learning and memory in insomnia rats, possibly by suppressing immunoinflammation through inhibition of the cGAS/STING signaling pathway.
3.Differences and Mechanisms of Combined Use of "Raw and Fried Ziziphi Spinosae Semen" in Anmeidan and Its Disassembled Prescriptions in Improving Anxiety and Cognitive Impairment in Insomnia Rat Model Based on Serum Metabolomics
Kang SUN ; Bo XU ; Zijing YE ; Miao ZHU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(10):36-43
ObjectiveTo investigate the differences in efficacy and endogenous metabolic mechanisms of Anmeidan with combined use of raw and fried Ziziphi Spinosae Semen and its disassembled prescriptions in treating anxiety and cognitive impairment in insomnia rats. MethodsSixty rats were randomly divided into six groups (n=10 per group): blank group, model group, suvorexant group (30 mg·kg-1), Anmeidan group (9.09 g·kg-1), Anmeidan with absence of raw Ziziphi Spinosae Semen group (7.38 g·kg-1), and Anmeidan with absence of fried Ziziphi Spinosae Semen group (7.38 g·kg-1). An insomnia model was constructed by intraperitoneal injection of para-chlorophenylalanine (PCPA), followed by gavage administration of Anmeidan or its disassembled prescriptions. Anxiety levels were assessed using the open field test, while cognitive ability was evaluated via the novel object recognition test. The pathological morphology of hippocampal neurons was examined using electron microscopy. Serum samples were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for principal component analysis, metabolic profiling, identification of differential metabolites, and metabolic pathway analysis. ResultsCompared with the blank group, the model group exhibited significantly increased exercise mileage, exercise time, and the ratio of the number of entries into the peripheral zone to the total number of entries into both the peripheral and central zones exhibited a marked increase (P<0.05, P<0.01), while the novel object recognition index significantly decreased (P<0.05). Compared with the model group, the Anmeidan and suvorexant groups showed significantly reduced exercise mileage and exercise time (P<0.01). The ratio of the number of entries into the peripheral zone to the total number of entries into both the peripheral and central zones decreased (P<0.05), and a significant increase in the novel object recognition index (P<0.01). However, the disassembled prescription groups showed no significant improvement in open field test and novel object recognition test indices. Electron microscopy revealed that the Anmeidan group improved the pathological morphology of hippocampal neurons in insomnia rats. Metabolomics analysis identified 10 potential differential metabolites associated with Anmeidan's therapeutic effects, involving metabolic pathways related to phenylalanine and tryptophan biosynthesis and metabolism, as well as the serotonergic pathway. ConclusionThe combined use of raw and fried Ziziphi Spinosae Semen in Anmeidan is more effective than its disassembled prescriptions in alleviating anxiety and cognitive impairment in PCPA-induced insomnia rats. The underlying mechanism may be associated with metabolic pathways related to phenylalanine, tryptophan, and serotonin.
4.Effect of Anmeidan on Cognitive Function and Metabolic Profiling in Insomnia Model Rats Based on Untargeted Metabolomics
Feizhou LI ; Bo XU ; Zijing YE ; Lianyu LI ; Andong ZHANG ; Ping WANG ; Linlin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(10):54-64
ObjectiveTo elucidate the potential mechanisms by which the classic prescription Anmeidan alleviates cognitive impairment in insomnia model rats through metabolic profiling. MethodsA total of 60 SD rats were randomly divided into six groups: blank group, model group, low-, medium-, and high-dose Anmeidan groups, and the Suvorexant group, with 10 rats in each group. Except for the blank group, the insomnia model was established in all other groups via intraperitoneal injection of para-chlorophenylalanine. The Suvorexant group was administered Suvorexant solution (30 mg·kg-1·d-1) by gavage, while the low-, medium-, and high-dose Anmeidan groups received Anmeidan decoction (4.55, 9.09, 18.18 g·kg-1·d-1) by gavage. The blank group received an equivalent volume of normal saline. The open field test was used to assess spatial exploration and anxiety/depressive-like behaviors in rats. Serum levels of epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), and vasoactive intestinal peptide (VIP) were measured using enzyme-linked immunosorbent assay (ELISA). Untargeted metabolomics was employed to identify differential metabolites in rat serum, and systematic biological methods were applied to analyze the potential targets and pathways of Anmeidan. ResultsCompared to the blank group, the model group exhibited significant reductions in total distance traveled, average speed, number of entries into the central area, time spent in the central area, and frequency of upright events (P<0.01), along with significant decreases in VIP, EGF, and BDNF levels (P<0.05,P<0.01). A total of 100 differential metabolites were identified between the model and blank groups. Compared to the model group, the low-, medium-, and high-dose Anmeidan groups showed significant increases in total distance traveled, average speed, number of entries into the central area, time spent in the central area, and frequency of upright events (P<0.05,P<0.01), as well as a significant increase in VIP levels (P<0.05,P<0.01). Anmeidan significantly reversed abnormal changes in 67 metabolites compared to the model group. A combined analysis identified 134 potential targets of Anmeidan, with network topology analysis suggesting that Caspase-3, B-cell lymphoma 2 (Bcl-2), nuclear transcription factor-κB (NF-κB), interleukin-1β (IL-1β), interleukin-2 (IL-2), matrix metalloproteinase-9 (MMP-9), and Toll-like receptor 4 (TLR4), among others, may serve as key targets of Anmeidan. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed major enriched pathways, including the cyclic adenosine monophosphate (cAMP) signaling pathway, hypoxia inducible factor-1 (HIF-1) signaling pathway, and IL-17 signaling pathway. ConclusionThis study demonstrates that Anmeidan can recalibrate abnormal metabolic profiles in insomnia model rats to mitigate cognitive impairment, with its mechanisms of action potentially involving the regulation of immune-inflammatory responses, energy metabolism, and apoptosis-related pathways.
5.Primary exploration of stage I anastomosis and T-tube fistulation in laparoscopic local resection of duodenal tumors
Lijie LUO ; Tao WANG ; Xinrui YE ; Xianzhe WANG ; Zhuoxuan ZHANG ; Zijing ZHANG ; Yaohui PENG ; Yan CHEN ; Haiping ZENG ; Haipeng TANG ; Jiantao LIN ; Weiqiang ZOU ; Wei WANG
Chinese Journal of Gastrointestinal Surgery 2025;28(2):198-202
Objective:To discuss the feasibility and safety of stage I anastomosis and T-tube fistulation in laparoscopic local resection of duodenal tumors.Methods:A descriptive case series study was used to retrospectively analyze the clinical diagnosis and treatment data of 14 patients with duodenal tumors who successfully underwent laparoscopic local resection of duodenal tumors + phase I anastomosis + T-tube ostomy in the Guangdong Provincial Hospital of Chinese Medicine and the First Affiliated Hospital of Guangzhou University of Chinese Medicine from October 2021 to March 2024. The resection and reconstruction steps of laparoscopic local resection of duodenal tumor + phase I anastomosis + T-tube ostomy are as follows: (1) after the safe margin is clear, the duodenal tumor is completely removed in full thickness, and the specimen bag is taken out and sent to frozen section to determine the nature of the tumor and the negative margin; (2) Perforate the anterior duodenal wall below the tumor plane, place a 16# T tube, and fix it with laparoscopic purse string suture. The abdominal wall is led out through the duodenum, and the duodenal T tube fistulation is performed; (3) The duodenum was continuously sutured in a full-thickness transverse shape, and the seromuscular layer was strengthened to form a phase I anastomosis. The nutritional improvement of patients after operation was mainly observed, and the intraoperative situation and postoperative complications were recorded.Results:No conversion to laparotomy, postoperative emergency reoperation, intraoperative and postoperative complications occurred in 14 patients with duodenal tumors who completed laparoscopic local resection of duodenal tumors + phase I anastomosis + T-tube ostomy. The operation time was (225.43 ± 56.54) min, and the intraoperative blood loss was (72.14 ± 74.65) ml. The patient recovered well after operation, and no severe postoperative abdominal bleeding occurred. Postoperative gastrointestinal angiography showed that the anastomotic stoma was unobstructed, and there were no stenosis, anastomotic leakage and other related complications. There was no significant difference in serum albumin [(37.09 ± 3.53) g/L vs. (37.52 ± 4) g/L] and hemoglobin [(100.79 ± 31.93) g/L vs. (103.07 ± 19.6) g/L] between before and 1 week after operation ( P > 0.05). Conclusion:Laparoscopic local resection of duodenal tumor + phase I anastomosis + T-tube fistulation can be used as one of the safe and feasible improved methods for local resection of duodenal tumor to effectively reduce the occurrence of related complications.
6.Anmeidan Regulates Hepatic Neurotransmitters and Circadian Rhythm in Rat Model of Insomnia via OX1R/PLCβ-1/PKCα/ERK1/2 Signaling Pathway
Bo XU ; Ping WANG ; Jing XIA ; Guangjing XIE ; Zijing YE ; Qinghua QIN ; Jing CHENG
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(15):11-20
ObjectiveTo explore the effect and mechanism of the classic famous prescription Anmeidan (AMD) developed in the Qing Dynasty in regulating the hepatic neurotransmitters and circadian rhythm in the rat model of insomnia via the orexin-1 receptor (OX1R)/phosphatidylinositol-specific phospholipase Cβ-1 (PLCβ-1)/protein kinase Cα (PKCα)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. MethodSixty SPF-grade SD rats were randomized into blank, model, suvorexant (30 mg·kg-1·d-1), and low-, medium-, and high-dose (4.55, 9.09, 18.09 g·kg-1·d-1, respectively) AMD groups, with 10 rats in each group. The rats in other groups except the blank group were modeled by intraperitoneal injection of p-chlorophenylalanine (PCPA) and administrated with corresponding drugs by gavage, and the blank group received an equal volume of normal saline. The general condition, body mass, and 24 h autonomic activity of each group were observed. The pathological changes of the liver tissue were observed by hematoxylin-eosin(HE)staining and Masson staining. The expression of gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), epinephrine (EPI), norepinephrine (NE), and acetylcholine (ACh) in the liver tissue was detected by enzyme-linked immunosorbent assay. The glutamate (Glu) expression in the liver tissue was detected by the biochemical method. The mRNA levels of biological clock genes Per1, Per2, Cry1, Cry2, Bmal1, and Bmal2 in the liver were determined by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). The protein and mRNA levels of factors in the OX1R/PLCβ-1/PKCα/ERK1/2 signaling pathway in the liver were determined by Western blot and Real-time PCR, respectively. ResultCompared with the blank group, the modeling decreased the body mass (P<0.05, P<0.01) and caused mania and disturbed resting rhythms (P<0.01), hepatic muscle fiber fracture, and edema with inflammatory cell infiltration. In addition, the modeling decreased the GABA, 5-HT, EPI, NE, and ACh content, increased Glu content (P<0.01), down-regulated the mRNA levels of Per1, Per2, Cry1, and Cry2 (P<0.01), up-regulated the mRNA levels of Bmal1 and Bmal2 (P<0.01), and promoted the expression of OX1R, PLCβ-1, PKCα, and ERK1/2 at both protein and mRNA levels (P<0.01). Compared with the model group, suvorexant and AMD increased the body mass (P<0.05, P<0.01), alleviated the mania, and increased the resting time and frequency (P<0.05, P<0.01). Moreover, the medications elevated the levels of GABA, 5-HT, EPI, NE, and ACh, lowered the Glu level, up-regulated the mRNA levels of Per1, Per2, Cry1, and Cry2 (P<0.05, P<0.01), down-regulated the mRNA levels of Bmal1 and Bmal2, and inhibited the expression of OX1R, PLCβ-1, PKCα, and ERK1/2 at both mRNA and protein levels (P<0.05, P<0.01). ConclusionAMD can regulate hepatic neurotransmitters and improve circadian rhythm in insomniac rats by inhibiting the OX1R/PLCβ-1/PKCα/ERK1/2 signaling pathway, and high-dose AMD demonstrated the strongest effect.
7.Application of anterior esophageal wall full layer fixation and gastric tube guidance in total laparoscopic overlap method for intracorporeal esophagojejunostomy
Yan CHEN ; Xinrui YE ; Lijie LUO ; Zijing ZHANG ; Wenjun XIONG ; Haigang YANG ; Yaohui PENG ; Zeyu LIN ; Zhuoxuan ZHANG ; Wei WANG
Chinese Journal of Gastrointestinal Surgery 2024;27(10):1074-1079
Objective:To explore the application of anterior esophageal wall full layer fixation and gastric tube guidance in total laparoscopic overlap method for intracorporeal esophagojejunostomy.Methods:Overlap esophagojejunostomy with anterior esophageal wall full layer fixation and gastric tube guidance is suitable for patients with advanced gastric cancer (clinical stage: cT1b~4aN0~3M0) and esophageal invasion <3 cm, who underwent radical total gastrectomy+ overlap esophagojejunostomy. The main operation procedure was performed as follows: A titanium clip was used for fixation of the full anterior wall of esophagus before overlap esophagojejunostomy, and the side‐to‐side esophagojejunostomy was performed with the linear stapler under the guidance of gastric tube. Then the titanium clip was removed after confirming that the correct cavity was entered. Finally, the common outlet was closed by two barbed sutures. A descriptive case series study was conducted. The clinical data of patients who underwent laparoscopic radical gastrectomy and overlap esophagojejunostomy with anterior esophageal wall full layer fixation and gastric tube guidance in Guangdong Provincial Hospital of Chinese medicine and the First Affiliated Hospital of Guangzhou University of Chinese medicine from May 2021 to June 2023 were retrospectively analyzed.Results:A total of 42 patients were collected, and all of them were successfully completed laparoscopic total radical gastrectomy without conversion to laparotomy or perioperative death. The esophagojejunostomy time, operative time, intraoperative blood loss was 17(5‐25) minutes, (258.8±38.0) minutes and 50(20‐200) ml, respectively. The incidence of esophageal false lumen was 0%, and there were no intraoperative complications. The time of gastric tube removal, initial fluid diet intake and the duration of postoperative hospital were 2(1‐5) , 4(1‐8) and 8(4‐21) days, respectively. There were no postoperative anastomotic hemorrhage, anastomotic stenosis and other related complications. One patient (2.38%) developed a Clavien‐Dindo IIIb complication, which was abdominal hemorrhage after operation. The second surgical exploration confirmed that the patient was bleeding due to gastroduodenal artery rupture. After intraoperative suture hemostasis, fluid expansion, blood transfusion and other treatments, the patient was discharged on the 15th day after the operation. Three patients (7.14%) developed Clavien‐Dindo grade II complications, including anastomotic leakage, chylous leakage and pulmonary infection, and were discharged after conservative treatment such as anti‐infection and prolonged retention of drainage tube.Conclusions:Laparoscopic overlap method for intracorporeal esophagojejunostomy with anterior esophageal wall fixation and gastric tube guidance can shorten the time of esophagojejunostomy and prevent the occurrence of false lumen, and do not increase anastomose‐related complications.
8.Application of anterior esophageal wall full layer fixation and gastric tube guidance in total laparoscopic overlap method for intracorporeal esophagojejunostomy
Yan CHEN ; Xinrui YE ; Lijie LUO ; Zijing ZHANG ; Wenjun XIONG ; Haigang YANG ; Yaohui PENG ; Zeyu LIN ; Zhuoxuan ZHANG ; Wei WANG
Chinese Journal of Gastrointestinal Surgery 2024;27(10):1074-1079
Objective:To explore the application of anterior esophageal wall full layer fixation and gastric tube guidance in total laparoscopic overlap method for intracorporeal esophagojejunostomy.Methods:Overlap esophagojejunostomy with anterior esophageal wall full layer fixation and gastric tube guidance is suitable for patients with advanced gastric cancer (clinical stage: cT1b~4aN0~3M0) and esophageal invasion <3 cm, who underwent radical total gastrectomy+ overlap esophagojejunostomy. The main operation procedure was performed as follows: A titanium clip was used for fixation of the full anterior wall of esophagus before overlap esophagojejunostomy, and the side‐to‐side esophagojejunostomy was performed with the linear stapler under the guidance of gastric tube. Then the titanium clip was removed after confirming that the correct cavity was entered. Finally, the common outlet was closed by two barbed sutures. A descriptive case series study was conducted. The clinical data of patients who underwent laparoscopic radical gastrectomy and overlap esophagojejunostomy with anterior esophageal wall full layer fixation and gastric tube guidance in Guangdong Provincial Hospital of Chinese medicine and the First Affiliated Hospital of Guangzhou University of Chinese medicine from May 2021 to June 2023 were retrospectively analyzed.Results:A total of 42 patients were collected, and all of them were successfully completed laparoscopic total radical gastrectomy without conversion to laparotomy or perioperative death. The esophagojejunostomy time, operative time, intraoperative blood loss was 17(5‐25) minutes, (258.8±38.0) minutes and 50(20‐200) ml, respectively. The incidence of esophageal false lumen was 0%, and there were no intraoperative complications. The time of gastric tube removal, initial fluid diet intake and the duration of postoperative hospital were 2(1‐5) , 4(1‐8) and 8(4‐21) days, respectively. There were no postoperative anastomotic hemorrhage, anastomotic stenosis and other related complications. One patient (2.38%) developed a Clavien‐Dindo IIIb complication, which was abdominal hemorrhage after operation. The second surgical exploration confirmed that the patient was bleeding due to gastroduodenal artery rupture. After intraoperative suture hemostasis, fluid expansion, blood transfusion and other treatments, the patient was discharged on the 15th day after the operation. Three patients (7.14%) developed Clavien‐Dindo grade II complications, including anastomotic leakage, chylous leakage and pulmonary infection, and were discharged after conservative treatment such as anti‐infection and prolonged retention of drainage tube.Conclusions:Laparoscopic overlap method for intracorporeal esophagojejunostomy with anterior esophageal wall fixation and gastric tube guidance can shorten the time of esophagojejunostomy and prevent the occurrence of false lumen, and do not increase anastomose‐related complications.
9.Anti-bacterial effect and its mechanism of lavender essential oil against multi-drug resistant Acinetobacter baumannii
Man ZHAO ; Zijing WU ; Cun SUN ; Yan YE ; Ting CHEN ; Shulin LIU ; Baohang ZHU ; Anni ZHAO ; Zhen SONG ; Yun YANG ; Hongwu SUN ; Hao ZENG
Journal of Army Medical University 2024;46(18):2046-2056
Objective To investigate the antibacterial effect and its preliminary mechanism of lavender essential oil on multi-drug resistant Acinetobacter baumannii.Methods Micro-dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)of lavender essential oil against multi-drug resistant Acinetobacter baumannii,and bactericidal kinetic study was employed to determine the onset and maintenance time of lavender essential oil.Meanwhile,the promoting and therapeutic effects of lavender essential oil on wound healing were observed in a mouse model of infection.Subsequently,crystal violet staining was used to determine the inhibition and clearance of multi-drug resistant Acinetobacter baumannii biofilm by lavender essential oil,and laser confocal microscopy was utilized to observe the survival of bacteria in biofilms.NanoDrop instrument was utilized to quantify the leakage of bacterial DNA nucleic acid and protein after intervention with 3 and 6 mg/mL lavender essential oil,and the leakage of bacterial potassium ion was measured by potassium ion test kit.Proteomics technology combined with bio-informatics were applied to explore the action mechanism of lavender essential oil against multi-drug resistant Acinetobacter baumannii.Results The MIC and MBC of lavender essential oil were both 6 mg/mL,which could kill almost all multi-drug resistant Acinetobacter baumannii at the time point of 120 min,and showed an obvious dose-and time-dependent manner.The overall animal model evaluation showed that both 3 and 6 mg/mL lavender essential oil could promote wound healing,and the curative effect was obvious.Further studies confirmed that 3 mg/mL lavender essential oil had a certain biofilm inhibitory effect on multi-drug resistant Acinetobacter baumannii,and 6 mg/mL also had a certain biofilm clearance effect under the same conditions.Meanwhile,when incubated at 37℃ for 1 h,the dose of 3 mg/mL could increase the leakage of DNA nucleic acid and protein,and significantly promote the efflux of potassium ions.Proteomic analysis suggested that the antibacterial effect of lavender essential oil may be related to affecting the oxidorereductase activity and cell metabolic process of multi-drug resistant Acinetobacter baumannii,and interfering with the biosynthesis of cell wall/membrane/envelope and other structures.Conclusion Lavender essential oil at 3 mg/mL can play an antibacterial effect against multi-drug resistant Acinetobacter baumannii,and its mechanism may be related to the destruction of bacterial biofilm and interference with bacterial metabolism.
10.Study on the Mechanism of AMD Regulating Orexin Signaling Pathway to Improve Learning and Memory of SD zebrafish Model
Zijing YE ; Bo XU ; Jing XIA ; Haifei LU ; Yumeng CHEN ; Mengying HUANG ; Ping WANG
World Science and Technology-Modernization of Traditional Chinese Medicine 2024;26(3):731-741
Objective To observe the effects of AnMeiDan(AMD),a representative formula of Peiyuan Guben Tranquilisation method,on the learning memory of zebrafish model of sleep deprivation(SD),and to explore the possible mechanism and the optimal apparent dose based on the Orexin signalling pathway.Methods 120 4-month-old wild-type AB line zebrafish were randomly divided into blank group,model group,AMD low-dose group(0.009 mg·mL-1·d-1),AMD medium-dose group(0.027 mg·mL-1·d-1),AMD high-dose group(0.081 mg·mL-1·d-1),and melatonin group(0.4 mg·mL-1·d-1).Zebrafish SD model was established by LED light induction method,24 h behavioural changes of zebrafish in each group were monitored by zebrafish behavioural tracking system,differences in learning and memory ability of zebrafish in each group were detected by T-maze,the state and number of neurons in the brain of zebrafish in each group were observed by Hematoxylin-Eosin(HE)staining method,the structural changes of neurons in the brain of zebrafish in each group were observed by transmission electron microscopy,and changes in neuron structure in the brain of zebrafish in each group were detected by ELISA method.The levels of Orexin A(OXA)and Orexin B(OXB)in zebrafish were detected by enzyme-linked immunosorbent assay(ELISA),and the protein and mRNA expression levels of OXA/p38 mitogen-activated protein kinase(P38 MAPK)/extracellular regulated protein kinase(ERK1/2)were detected by protein immunoblotting and real-time fluorescence quantitative assay(RT-PCR)in zebrafish.Results Compared with the blank group,the total number of rests,duration and distance were reduced in the model group(P<0.05),and the latency to enter the target region(OR)was prolonged(P<0.01);the number of neuronal cells was reduced,the nuclei were degenerated(P<0.01),cytoplasmic hyalinosis was increased(P<0.01),and light colouring was observed,the nuclei were collapsed,the nuclear membranes were ruptured and lysed,the chromatin was solidified,and the mitochondria were swollen and deformed with internal cavities,and lamellipod-like myelin-like structure was formed;the brain tissue OXA,OXA,and ERK1/2 expression levels of the mRNAs and the mRNAs of these signaling pathways were increased(P<0.01).brain tissue OXA and OXB proteins were significantly elevated,OXA mRNA and protein were significantly up-regulated,and P38 MAPK and ERK1/2 mRNA and protein were significantly down-regulated(P<0.01).Compared with the model group,the drug intervention could prolong the total resting exercise time of SD zebrafish(P<0.05);shorten the latency of zebrafish to reach OR(P<0.05);protect the structure and morphology of neuronal cells,and alleviate the damage of brain tissues;reduce the content of OXA and OXB in the brain tissues(P<0.01);and down-regulate the expression of OXA mRNA and protein(P<0.01),and up-regulate the P38 MAPK and ERK1/2 mRNA and protein expression of P38 MAPK and ERK1/2(P<0.01);and the quantity-effect relationship was obvious,and the effect of high dose of AMD was optimal.Conclusion AMD can improve the learning and memory ability in zebrafish SD model,and its mechanism may be related to the regulation of OXA/P38 MAPK/ERK1/2 signalling pathway and alleviation of neuronal damage in the brain,and the high dose group of AMD showed the best effect.

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