Anxiety disorder is a major symptom of autism spectrum disorder (ASD) with a comorbidity rate of ~40%. However, the neural mechanisms of the emergence of anxiety in ASD remain unclear. In our study, we found that hyperactivity of basolateral amygdala (BLA) pyramidal neurons (PNs) in Shank3 InsG3680 knock-in (InsG3680^+/+) mice is involved in the development of anxiety. Electrophysiological results also showed increased excitatory input and decreased inhibitory input in BLA PNs. Chemogenetic inhibition of the excitability of PNs in the BLA rescued the anxiety phenotype of InsG3680^+/+ mice. Further study found that the diminished control of the BLA by medial prefrontal cortex (mPFC) and optogenetic activation of the mPFC-BLA pathway also had a rescue effect, which increased the feedforward inhibition of the BLA. Taken together, our results suggest that hyperactivity of the BLA and alteration of the mPFC-BLA circuitry are involved in anxiety in InsG3680^+/+ mice.
Animals ; Prefrontal Cortex/metabolism* ; Basolateral Nuclear Complex/metabolism* ; Mice ; Anxiety/metabolism* ; Nerve Tissue Proteins/genetics* ; Male ; Gene Knock-In Techniques ; Pyramidal Cells/physiology* ; Mice, Transgenic ; Neural Pathways/physiopathology* ; Mice, Inbred C57BL ; Microfilament Proteins

Objective: To explore the network structure of eating behaviors with anxiety, depression and perceived stress in adolescents, so as to provide a basis for effective prevention and intervention of eating behavior problems and negative emotions in adolescents. Methods: Based on the Psychology and Behavior Investigation of Chinese Residents (2021) database, the study was conducted among 3 087 adolescents. Sakata Eating Behavior Scale Short From(EBS-SF) was used to investigate their eating behaviors. The Patient Health Questionnaire-9(PHQ-9), Generalized Anxiety Disorder Scale-7 Item(GAD-7), and Perceived Stress Questionnaire-3 Item (PSQ-3) were used to evaluate their depression, anxiety and perceived stress. Network analysis method was applied to construct a network of eating behaviors and negative emotional symptoms among adolescents, so as to evaluate the centrality, bridge strength, stability and accuracy of each item. Results: The total scores of eating behaviors, depression,anxiety and stress perception in adolescents were 17.41±4.53,6.95±6.08,4.86±5.03,9.34±3.80,respectively. The symptom with the highest intensity and expected impact was "I am only satisfied when I buy more food than I need", with a node intensity and expected impact value of 4.37. The nodes Depression and Anxiety were the most closely connected(weight=0.87). There were no statistically significant differences in the network structure( M =0.13,0.11) and network connection strength(female and male:4.16,4.06, s =0.10;urban and rural areas:4.08,4.07, s =0.01) between different sexes and residents ( P >0.05). Conclusion The negative impact of comorbidities such as anxiety, depression, perceived stress and eating behaviors among adolescents can be reduced through targeted prevention and intervention of core symptoms and bridging symptoms.

This study was aimed at identifying the pathogenic bacteria responsible for the death of a young tiger at the Fujian Meihua Mountain South China Tiger Breeding Research Institute.Tissue samples from the lungs,liver,and intestines of the deceased tiger were collected,and the bacteria were cultured inasterile environment.The bacterial strains were characterized according to their morphological and molecular biological properties,including assessment of virulence genes and antibiotic resistance genes,mouse lethality tests,and antibiotic susceptibility evaluations.A predominant bacterial strain isolated from the liver of the deceased tiger was identified as enterotoxigenic Escherichia coli(ETEC)strain Tiger22513F.Phylogenetic analysis of the 16S rRNA gene revealed that the Tiger22513F strain exhibited close genetic similarity to the reference strain ETEC(MF919609.1),with 99.9%nucleotide similarity,and resided on the same evolutionary branch.The Tiger22513F strain contained 11 antibiotic resistance genes(tetA,sul1,sul3,cmlA,floR,blaTEM,blaSHV,blaCMY-2,qnrA,qnrS,and qnrD)along with five virulence genes(VT1,fyuA,tsh,iucD,and ST).Mouse lethality tests indicated significant pathogenicity toward mice,affecting primarily the lungs,liver,and intestines.Antibiotic susceptibility testing demonstrated that this strain exhibited resistance to various classes of beta-lactam antibiotics,as well as quinolones and aminoglycosides.This investigation successfully isolated a multi-drug resistant enterotoxigenic Escherichia coli strain with pronounced pathogenicity from the liver of a deceased tiger;thus providing valuable scientific insights for clinical diagnosis,as well as prevention and control measures,against ETEC infections in South China tigers.

OBJECTIVE To study the safety of Shuangshu decoction in rats and its efficacy in improving functional dyspepsia （FD） in rats. METHODS In safety test， 40 rats were divided into blank control group， Shuangshu decoction low-dose， medium- dose and high-dose groups [108， 216， 324 g/（kg·d）， calculated by raw medicine， the same applies below]； they were given relevant medicine intragastrically， for continuous 14 days. The mortality and toxic reactions of rats were recorded， and the organ indexes of the liver， kidney， spleen， lung and heart of rats were calculated； the pathological morphological changes in the liver， kidney， spleen， lung， heart， stomach， duodenum， and colon were observed to evaluate the acute toxicity of Shuangshu decoction. Another 40 rats were grouped and administered in the same way for 30 consecutive days. The mortality and toxic reactions of the rats were recorded， and the corresponding organ indexes were calculated. The pathological morphological changes in the corresponding organs were observed， and blood routine and serum biochemical indicators were measured， in order to assess the subacute toxicity of Shuangshu decoction. In pharmacodynamic experiments: 50 rats were divided into blank control group， model group， and Shuangshu decoction low-， medium-， and high-dose groups （9.45， 18.9， 37.8 g/kg）， with 10 rats in each group. Except for blank control group， rats in all other groups were used to establish the FD rat model by subcutaneous injection of loperamide （3.5 mg/kg）. Rats in each group were administered the corresponding drug solution/normal saline intragastrically， once a day， for 14 consecutive days. After the last medication， fecal moisture content， intestinal propulsion rate， gastric emptying rate and serum level of motilin were all detected， and interstitial cell of Cajal （ICC） ultrastructure of rats was observed in colon tissue. RESULTS The safety experiments showed that no death occurred in each dose group， and no significant difference was found in organ coefficient， routine blood and serum biological index， compared to blank control group （P＞0.05）； no abnormality was found in organ appearance and pathological sections. The results of the pharmacodynamic experiments showed that， compared with the blank control group， the fecal moisture content， gastric emptying rate， intestinal propulsion rate， and serum motilin levels in the model group were significantly decreased （P＜0.05）； in the colonic tissue， the mitochondria in the ICC exhibited severe swelling with the disappearance of cristae， and the endoplasmic reticulum was dilated. Compared with model group， the rats in Shuangshu decoction high-dose group showed significant increases in the above quantitative indicators （P＜ 0.05）； additionally， there was a large number of mitochondria in the ICC of the colonic tissue， with clear cristae and regular arrangement. CONCLUSIONS Shuangshu decoction is safe and has a beneficial improving effect on FD rats； its mechanism of action may be related to the regulation of gastrointestinal hormone expression to promote gastric emptying and intestinal propulsion， as well as the repair of mitochondrial structure in ICCs to restore gastrointestinal function.

Objective To observe the effects of Shenfu Injection on ferroptosis-related factors and oxidative stress-related indexes in rat cardiomyocytes treated with isoproterenol;To explore its mechanism in treating chronic heart failure.Methods Isoproterenol was used to induce rat H9c2 cardiomyocyte injury.The cells were divided into normal group,model group,Shenfu Injection group and Ferrostatin-1 group,and treated with corresponding intervention.Transmission electron microscopy was used to observe the ultrastructure of cellular mitochondria,ferrous ion fluorescent probe was used to detect Fe2+content in cells,flow cytometry was used to detect intracellular contents of ROS and Lipid-ROS;colorimetry was used to detect the contents of MDA,GSH,SOD and GSH-Px in cells;Western blot and RT-qPCR were used to detect the protein and mRNA expressions of GPX4,FTH1,SLC7A11,p53,COX2 and Nrf2 in cells.Results Compared with the normal group,the survival rate of H9c2 cells in the model group was significantly reduced(P<0.01),with cell swelling and rupture,mitochondrial shrinkage,decreased quantity and disordered arrangement,more damaged mitochondria,the contents of Fe2+,ROS,Lipid-ROS and MDA in cells significantly increased(P<0.01),while the contents of GSH,SOD and GSH-Px significantly decreased(P<0.01),the expressions of p53,COX2 protein and mRNA significantly increased(P<0.01),the expressions of GPX4,FTH1,SLC7A11 and Nrf2 protein and mRNA significantly decreased(P<0.01).Compared with the model group,the survival rates of H9c2 cells in Shenfu Injection group and Ferrostatin-1 group significantly increased(P<0.01),there was a larger number of normal mitochondria and a more complete structure,the contents of Fe2+,ROS,Lipid-ROS and MDA in cells were significantly decreased(P<0.01),while the contents of GSH,SOD and GSH-Px were significantly increased(P<0.05,P<0.01),the expressions of p53,COX2 protein and mRNA were significantly decreased(P<0.05,P<0.01),while the expressions of GPX4,FTH1,SLC7A11 and Nrf2 protein and mRNA significantly increased(P<0.01).Conclusion Shenfu Injection can reduce p53 expression,weaken its inhibitory effect on SLC7A11,thereby promoting GPX4 expression,inhibiting ferroptosis,reducing lipid peroxide accumulation,increasing cellular antioxidant capacity,and alleviating myocardial cell oxidative damage.

This study was aimed at identifying the pathogenic bacteria responsible for the death of a young tiger at the Fujian Meihua Mountain South China Tiger Breeding Research Institute.Tissue samples from the lungs,liver,and intestines of the deceased tiger were collected,and the bacteria were cultured inasterile environment.The bacterial strains were characterized according to their morphological and molecular biological properties,including assessment of virulence genes and antibiotic resistance genes,mouse lethality tests,and antibiotic susceptibility evaluations.A predominant bacterial strain isolated from the liver of the deceased tiger was identified as enterotoxigenic Escherichia coli(ETEC)strain Tiger22513F.Phylogenetic analysis of the 16S rRNA gene revealed that the Tiger22513F strain exhibited close genetic similarity to the reference strain ETEC(MF919609.1),with 99.9%nucleotide similarity,and resided on the same evolutionary branch.The Tiger22513F strain contained 11 antibiotic resistance genes(tetA,sul1,sul3,cmlA,floR,blaTEM,blaSHV,blaCMY-2,qnrA,qnrS,and qnrD)along with five virulence genes(VT1,fyuA,tsh,iucD,and ST).Mouse lethality tests indicated significant pathogenicity toward mice,affecting primarily the lungs,liver,and intestines.Antibiotic susceptibility testing demonstrated that this strain exhibited resistance to various classes of beta-lactam antibiotics,as well as quinolones and aminoglycosides.This investigation successfully isolated a multi-drug resistant enterotoxigenic Escherichia coli strain with pronounced pathogenicity from the liver of a deceased tiger;thus providing valuable scientific insights for clinical diagnosis,as well as prevention and control measures,against ETEC infections in South China tigers.

Objective To observe the effects of Shenfu Injection on ferroptosis-related factors and oxidative stress-related indexes in rat cardiomyocytes treated with isoproterenol;To explore its mechanism in treating chronic heart failure.Methods Isoproterenol was used to induce rat H9c2 cardiomyocyte injury.The cells were divided into normal group,model group,Shenfu Injection group and Ferrostatin-1 group,and treated with corresponding intervention.Transmission electron microscopy was used to observe the ultrastructure of cellular mitochondria,ferrous ion fluorescent probe was used to detect Fe2+content in cells,flow cytometry was used to detect intracellular contents of ROS and Lipid-ROS;colorimetry was used to detect the contents of MDA,GSH,SOD and GSH-Px in cells;Western blot and RT-qPCR were used to detect the protein and mRNA expressions of GPX4,FTH1,SLC7A11,p53,COX2 and Nrf2 in cells.Results Compared with the normal group,the survival rate of H9c2 cells in the model group was significantly reduced(P<0.01),with cell swelling and rupture,mitochondrial shrinkage,decreased quantity and disordered arrangement,more damaged mitochondria,the contents of Fe2+,ROS,Lipid-ROS and MDA in cells significantly increased(P<0.01),while the contents of GSH,SOD and GSH-Px significantly decreased(P<0.01),the expressions of p53,COX2 protein and mRNA significantly increased(P<0.01),the expressions of GPX4,FTH1,SLC7A11 and Nrf2 protein and mRNA significantly decreased(P<0.01).Compared with the model group,the survival rates of H9c2 cells in Shenfu Injection group and Ferrostatin-1 group significantly increased(P<0.01),there was a larger number of normal mitochondria and a more complete structure,the contents of Fe2+,ROS,Lipid-ROS and MDA in cells were significantly decreased(P<0.01),while the contents of GSH,SOD and GSH-Px were significantly increased(P<0.05,P<0.01),the expressions of p53,COX2 protein and mRNA were significantly decreased(P<0.05,P<0.01),while the expressions of GPX4,FTH1,SLC7A11 and Nrf2 protein and mRNA significantly increased(P<0.01).Conclusion Shenfu Injection can reduce p53 expression,weaken its inhibitory effect on SLC7A11,thereby promoting GPX4 expression,inhibiting ferroptosis,reducing lipid peroxide accumulation,increasing cellular antioxidant capacity,and alleviating myocardial cell oxidative damage.

Objective To establish a method for the simultaneous determination of DOX·HCl and LND. Methods HPLC was performed on Agilent 5 HC-C₁₈（2） （4.6 mm × 250 mm, 5 µm） column. The mobile phase was methanol-0.1% TFA aqueous solution, and the gradient elution procedure were: 0 to 3 min, 65% methanol; 3 to 7 min, 65%→90% methanol; 7 to 13 min, 90% methanol; 13 to 15 min, 90%→65% methanol; 15 to 20 min, 65% methanol. The collection time was 20 min, the balance time was 3 min, the UV detection wavelengths were 205 nm and 253 nm. The flow rate was 1.0 ml/min and the column temperature was 35℃. The amount of inlet was 10 µl. Results The method was highly specific, and both DOX·HCl and LND exhibited good linearity in the concentration range of 1-40 µg/ml and 6-240 µg/ml, respectively. The two compounds’ precision, stability, and recovery satisfied the requirements of the method. Conclusion This study established a HPLC method that was suitable for the simultaneous detection of DOX·HCl and LND. This method’s high level of specificity, accuracy, and reliability .

ObjectiveTo investigate the effect and possible mechanism of Shenfu Injection （参附注射液） on rat cardiomyocyte injury induced by isoproterenol from the perspective of regulating the high mobility group protein B1 （HMGB1）/ Toll-like receptor 4 （TLR4）/nuclear factor κB （NF-κB） pathway through the deacetylation modification of silent information regulator 1 （SIRT1）. MethodsThe optimal concentration and intervention duration of isoproterenol hydrochloride and the optimal intervention concentration of Shenfu Injection were screened out by CCK-8 method. Logarithmically growing H9c2 rat cardiomyocytes were taken at 5×10⁴ cells/well and divided into normal group， model group， Shenfu Injection group， and SIRT1 inhibitor group， with 3 replicates in each group.Except for the normal group， the cells in the other groups were induced by isoproterenol hydrochloride to establish a chronic heart failure cell model. After modeling， the Shenfu Injection group was given Shenfu Injection at the optimal intervention concentration， and the SIRT1 inhibitor group was given 1 μmol/L of SIRT1 inhibitor EX-527， for optimal intervention duration.CCK-8 assay was used to detect the cell activity and calculate the inhibitory rate. ELISA assay was used to detect the nicotinamide adenine dinucleotide oxidation state/ nicotinamide adenine dinucleotide reduction state （NAD+/NADH） in cardiomyocytes. Immunofluorescence was used to detect the immunofluorescence localization of HMGB1 and SIRT1 in cardiomyocytes. Western blotting was used to detect the protein expression of acetylated HMGB1 in cardiomyocytes， HMGB1 in the nucleus and cytoplasm， and SIRT1， TLR4， myeloid differentiation factor 88 （MYD88） and NF-κB p65 in cardiomyocytes. RT-qPCR was used to detect the mRNA expression of SIRT1， HMGB1， TLR4， MYD88 and NF-κB p65 in cardiomyocytes. ResultsThe optimal intervention concentration of isoproterenol hydrochloride was 300 μmol/L， and the intervention duration was 48 hours； 8% was the optimal intervention concentration of Shenfu Injection. Compared to those in the normal group， the cell activity， NAD+/NADH value， nuclear HMGB1 protein expression， cardiomyocyte SIRT1 protein and mRNA expression in the model group decreased， while the cell inhibition rate， cardiomyocyte acetylated HMGB1 and cytoplasmic HMGB1 protein expression， cardiomyocyte TLR4， MYD88， NF-κB p65 protein and mRNA expression all increased （P＜0.05）； fluorescence localization showed that the content of HMGB1 in cardiomyocytes in the model group increased and was localized in both the nucleus and cytoplasm.Compared to the model group and the SIRT1 inhibitor group， the Shenfu Injection group showed significant improvements in all the above indicators （P＜0.05）； fluorescence localization showed that the SIRT1 content increased in the Shenfu injection group， while the HMGB1 content decreased， and was mainly located in the nucleus. ConclusionShenfu Injection can improve myocardial cell damage by increasing SIRT1 expression to reduce the acetylation level of HMGB1， regulating the HMGB1/TLR4/NF-κB pathway and inhibiting the nuclear translocation of HMGB1.