ObjectiveTo investigate the mechanism of Zuogui Wan （ZGW） in improving ovarian inflammatory microenvironment and stemness of ovarian germline stem cells （OSCs） for treating ovarian aging via the cyclic guanosine monophosphate/adenosine monophosphate synthase （cGAS）/stimulator of interferon genes （STING） signaling pathway. MethodsForty C57BL/6 female mice were randomly divided into a blank group， a model group， a low-dose ZGW group （2.7 g·kg^-1）， a high-dose ZGW group （5.4 g·kg^-1）， and an estradiol valerate group （0.15 mg·kg^-1）， with 8 mice in each group. Except the blank group， all other groups received a single intraperitoneal injection of cyclophosphamide at 120 mg·kg^-1 to establish an ovarian aging mouse model. After successful modeling， each group was continuously administered for 4 weeks， once daily. The physiological status of the mice was observed， and the ovarian index was calculated. The estrus cycle of the mice was monitored. Hematoxylin-eosin （HE） staining was used to observe pathological changes in ovarian tissue. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum sex hormone levels. Serum inflammatory factors interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and mouse interleukin-6 （IL-6） levels were detected using kits. Western blot was used to detect the protein expression of ovarian cGAS， STING， p-STING， TANK-binding kinase 1 （TBK1）， p-TBK1， interferon-induced transmembrane protein 3 （Fragilis）， and Vasa homolog protein （MVH）. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of inflammatory factors in ovarian tissue. Immunofluorescence double labeling was performed to locate OSCs in ovarian tissues， and fluorescence intensities of OSCs markers MVH and octamer binding transcription factor 4 （Oct4） were calculated. ResultsCompared with the blank group， the model group showed reduced body weight， ovarian wet weight， and ovarian index （P<0.01） and a disordered estrus cycle （P<0.01）. In addition， the levels of serum follicle-stimulating hormone （FSH）， TNF-α， IL-6， and IL-1β were increased （P<0.01）， while anti-Müllerian hormone （AMH） and estradiol （E₂） levels were decreased （P<0.01）. The protein expression of cGAS， p-STING/STING， and p-TBK1/TBK1 in ovarian tissue was increased （P<0.05， P<0.01）， while that of OSCs stemness factors MVH and Fragilis was reduced （P<0.01）. Immunofluorescence indicated a reduction in MVH and Oct4 expression in OSCs （P<0.01）. The mRNA expression of inflammatory factors TNF-α， IL-6， and IL-1β in ovarian tissue was increased （P<0.05， P<0.01）. Compared with the model group， the treatment groups exhibited improved body weight， ovarian wet weight， and ovarian index （P<0.05） and a reduced rate of estrus cycle disorder （P<0.05， P<0.01）. The levels of serum FSH， TNF-α， IL-6， and IL-1β were decreased （P<0.05， P<0.01）， while AMH and E₂ levels were increased （P<0.01）. The protein expression levels of cGAS， p-STING/STING， and p-TBK1/TBK1 in ovarian tissue were decreased （P<0.05）， while the protein expression of MVH and Fragilis was increased （P<0.05）， and the fluorescence intensities of MVH and Oct4 were increased （P<0.05， P<0.01）. The mRNA expression of inflammatory factors in ovarian tissue was decreased （P<0.05）. ConclusionZGW alleviate ovarian inflammatory response， regulate ovarian microenvironment homeostasis， and maintain stemness of OSCs in ovarian aging mice probably by modulating the cGAS-STING signaling pathway， thereby improving ovarian function and delaying ovarian aging.

ObjectiveTo explore the effect of serum containing Zhenwutang on myocardial mast cell apoptosis induced by angiotensin Ⅱ （AngⅡ） and the mechanism of the correlation between apoptosis and mitochondrial autophagy. MethodsIn this experiment， AngⅡ and serum containing Zhenwutang with different concentrations were used to interfere with H9C2 cardiomyocytes for 24 h， and the survival rate of H9C2 cardiomyocytes was detected by cell counting kit-8 （CCK-8） to screen the optimal concentration for the experiment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the content of B-type natriuretic peptide （BNP） in cell culture supernatant， and immunofluorescence was used to detect the cell surface area to verify the construction of the myocardial mast cell model. Subsequently， the experiment was divided into a blank group （20% blank serum）， a model group （20% blank serum + 5×10^-5 mol·L^-1 AngⅡ）， low-， medium-， and high-dose （5%， 10% and 20%） serum containing Zhenwutang groups， an autophagy inhibitor group （1×10^-4 mol·L^-1 3-MA）， and autophagy inducer group （1×10^-7 mol·L^-1 rapamycin）. The apoptosis level of H9C2 cells and the changes of mitochondrial membrane potential were detected by flow cytometry. The lysosomal probe （Lyso Tracker） and mitochondrial probe （Mito Tracker） co-localization was employed to detect autophagy. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect Caspase-3， Caspase-9， B-cell lymphoma 2 （Bcl-2）， Bcl-2-related X protein （Bax）， and cytochrome C （Cyt C） in apoptosis-related pathways and the relative mRNA expression of ubiquitin ligase （Parkin）， phosphatase and tensin homolog （PTEN）-induced kinase 1 （PINK1）， and p62 protein in mitochondrial autophagy-related pathways. Western blot was used to detect cleaved Caspase-3， cleaved Caspase-9， Bax， Bcl-2， and Cyt C in apoptosis-related pathways， phosphorylated ubiquitin ligase （p-Parkin）， phosphorylated PTEN-induced kinase 1 （p-PINK1）， p62， and Bcl-2 homology domain protein Beclin1 in mitochondrial autophagy-related pathways， and the change of microtubule-associated protein 1 light chain 3 （LC3） Ⅱ/Ⅰ ratio. ResultsCCK-8 showed that when the concentration of AngⅡ was 5×10^-5 mol·L^-1， the cell activity was the lowest， and there was no cytotoxicity. At this concentration， the surface area of cardiomyocytes was significantly increased （P<0.01）， and the content of BNP in the supernatant of culture medium was significantly increased （P<0.05）. Therefore， AngⅡ with a concentration of 5×10^-5 mol·L^-1 was selected for the subsequent modeling of myocardial mast cells. Compared with the blank group， the model group and the autophagy inhibitor 3-MA group had a significantly increased apoptosis rate （P<0.01） and significantly decreased mitochondrial membrane potential （P<0.01）. The results of immunofluorescence co-localization showed that compared with the blank group， the model group had a significantly decreased number of red and green fluorescence spots. The results of Real-time PCR showed that compared with that in the blank group， the relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 in the model group was significantly up-regulated （P<0.01）， while the relative mRNA expression of Bcl-2， Parkin， and PINK1 was significantly down-regulated （P<0.01）. In addition， the relative protein expression of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 was significantly up-regulated （P<0.01）. The LC3Ⅱ/Ⅰ was significantly decreased， and the relative protein expression of Bcl-2， p-Parkin， p-PINK1， and Beclin1 was significantly down-regulated （P<0.01）. Compared with the model group， the serum containing Zhenwutang groups and the autophagy inducer group had significantly decreased apoptosis rate （P<0.01）， and the decrease ratio of mitochondrial membrane potential is significantly lowered （P<0.01） in a dose-dependent manner. Additionally， both red and green fluorescence spots became more in these groups. In the 3-MA group， the number of red and green fluorescence spots decreased significantly. The relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 was significantly down-regulated （P<0.05， P<0.01）， while that of Bcl-2， Parkin， and PINK1 was significantly up-regulated （P<0.01）. In the serum containing Zhenwutang groups， the relative protein expression levels of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 were significantly down-regulated （P<0.05，P<0.01）. The LC3Ⅱ/Ⅰ was significantly increased， and the relative protein expression levels of Bcl-2， p-Parkin， p-PINK1， and Beclin1 were significantly up-regulated （P<0.01）. ConclusionThe serum containing Zhenwutang can reduce the apoptosis of myocardial mast cells and increase mitochondrial autophagy. This is related to the inhibition of intracellular Bax/Bcl-2/Caspase-3 apoptosis pathway and regulation of Parkin/PINK1 mitochondrial autophagy pathway.

The innate immune system can be boosted in response to subsequent triggers by pre-exposure to microbes or microbial products, known as “trained immunity”. Compared to classical immune memory, innate trained immunity has several different features. Firstly, the molecules involved in trained immunity differ from those involved in classical immune memory. Innate trained immunity mainly involves innate immune cells (e.g., myeloid immune cells, natural killer cells, innate lymphoid cells) and their effector molecules (e.g., pattern recognition receptor (PRR), various cytokines), as well as some kinds of non-immune cells (e.g., microglial cells). Secondly, the increased responsiveness to secondary stimuli during innate trained immunity is not specific to a particular pathogen, but influences epigenetic reprogramming in the cell through signaling pathways, leading to the sustained changes in genes transcriptional process, which ultimately affects cellular physiology without permanent genetic changes (e.g., mutations or recombination). Finally, innate trained immunity relies on an altered functional state of innate immune cells that could persist for weeks to months after initial stimulus removal. An appropriate inducer could induce trained immunity in innate lymphocytes, such as exogenous stimulants (including vaccines) and endogenous stimulants, which was firstly discovered in bone marrow derived immune cells. However, mature bone marrow derived immune cells are short-lived cells, that may not be able to transmit memory phenotypes to their offspring and provide long-term protection. Therefore, trained immunity is more likely to be relied on long-lived cells, such as epithelial stem cells, mesenchymal stromal cells and non-immune cells such as fibroblasts. Epigenetic reprogramming is one of the key molecular mechanisms that induces trained immunity, including DNA modifications, non-coding RNAs, histone modifications and chromatin remodeling. In addition to epigenetic reprogramming, different cellular metabolic pathways are involved in the regulation of innate trained immunity, including aerobic glycolysis, glutamine catabolism, cholesterol metabolism and fatty acid synthesis, through a series of intracellular cascade responses triggered by the recognition of PRR specific ligands. In the view of evolutionary, trained immunity is beneficial in enhancing protection against secondary infections with an induction in the evolutionary protective process against infections. Therefore, innate trained immunity plays an important role in therapy against diseases such as tumors and infections, which has signature therapeutic effects in these diseases. In organ transplantation, trained immunity has been associated with acute rejection, which prolongs the survival of allografts. However, trained immunity is not always protective but pathological in some cases, and dysregulated trained immunity contributes to the development of inflammatory and autoimmune diseases. Trained immunity provides a novel form of immune memory, but when inappropriately activated, may lead to an attack on tissues, causing autoinflammation. In autoimmune diseases such as rheumatoid arthritis and atherosclerosis, trained immunity may lead to enhance inflammation and tissue lesion in diseased regions. In Alzheimer’s disease and Parkinson’s disease, trained immunity may lead to over-activation of microglial cells, triggering neuroinflammation even nerve injury. This paper summarizes the basis and mechanisms of innate trained immunity, including the different cell types involved, the impacts on diseases and the effects as a therapeutic strategy to provide novel ideas for different diseases.

BACKGROUND:Neuregulin 1 has been shown to be characterized in cell proliferation,differentiation,and vascular growth.Human amniotic mesenchymal stem cells are important seed cells in the field of tissue engineering,and have been shown to be involved in tissue repair and regeneration. OBJECTIVE:To construct human amniotic mesenchymal stem cells overexpressing neuregulin 1 and investigate their proliferation and migration abilities,as well as their effects on wound healing. METHODS:(1)Human amniotic mesenchymal stem cells were in vitro isolated and cultured and identified.(2)A lentivirus overexpressing neuregulin 1 was constructed.Human amniotic mesenchymal stem cells were divided into empty group,neuregulin 1 group,and control group,and transfected with empty lentivirus and lentivirus overexpressing neuregulin 1,or not transfected,respectively.(3)Edu assay was used to detect the proliferation ability of the cells of each group,and Transwell assay was used to detect the migration ability of the cells.(4)The C57 BL/6 mouse trauma models were constructed and randomly divided into control group,empty group,neuregulin 1 group,with 8 mice in each group.Human amniotic mesenchymal stem cells transfected with empty lentivirus or lentivirus overexpressing neuregulin-1 were uniformly injected with 1 mL at multiple local wound sites.The control group was injected with an equal amount of saline.(5)The healing of the trauma was observed at 1,7,and 14 days after model establishment.Histological changes of the healing of the trauma were observed by hematoxylin-eosin staining.The expression of CD31 on the trauma was observed by immunohistochemistry. RESULTS AND CONCLUSION:(1)Human amniotic mesenchymal stem cells overexpressing neuregulin-1 were successfully constructed.The mRNA and protein expression of intracellular neuregulin 1 was significantly up-regulated compared with the empty group(P<0.05).(2)The overexpression of neuregulin 1 promoted the migratory ability(P<0.01)and proliferative ability of human amniotic mesenchymal stem cells(P<0.05).(3)Human amniotic mesenchymal stem cells overexpressing neuregulin 1 promoted wound healing in mice(P<0.05)and wound angiogenesis(P<0.05).The results showed that overexpression of neuregulin 1 resulted in an increase in the proliferative and migratory capacities of human amniotic mesenchymal stem cells,significantly promoting wound healing and angiogenesis.

Pancreatitis is an inflammatory disease influenced by both environmental and genetic factors. It has a high prevalence and mortality rate worldwide, with no radical cure. Breakthroughs have been recently made in genetic research of pancreatitis. Susceptibility genes and pathways have been continuously discovered, which highlighted the roles of genetic factors in hypertriglyceridemic and chronic pancreatitis. Gene therapy may offer radical cure for pancreatitis, though it has remained at the research phase. This article has reviewed the genetic pathogenesis of pancreatitis and current status of gene therapy research, with an aim to provide a reference for attaining definitive cure for the disease.
Humans ; Genetic Therapy/methods* ; Pancreatitis/etiology* ; Genetic Predisposition to Disease ; Animals

AIM To investigate the repair effects of tauroursodeoxycholic acid(TUDCA)combined with bone marrow mesenchymal stem cells(BMSCs)transplantation on spinal cord injury(SCI)in rats.METHODS The rats were randomly divided into the sham operation group,the model group,the TUDCA group,the BMSCs transplantation group and the combination therapy of TUDCA and BMSCs transplantation group,with the SCI rat model established by Allen's method.The next day after modeling,the rats of TUDCA and combination therapy groups were given 200 mg/kg TUDCA by gavage.On the 3rd day after modeling,rats in BMSCs transplantation group and combination therapy group were injected with 1 mL tuned bone marrow BMSCs(the 3rd generation,1× 106/mL)via tail vein.Rats in the sham operation group and the model group were given gastric perfusion of normal saline and injection of 1 mL PBS through tail vein.On the 3rd,7th and 14th day after modeling,the rats had their motor function of hind limbs observed and BBB score determined.After the corresponding drug administration,the rats had their movement track of hind limbs recorded by footprint experiment;their the protein expressions of IL-6,IL-10,Arg-1,PI3K and Akt in spinal cord tissue detected by Western blot;their pathological changes of spinal cord tissue observed by HE staining and Nissl staining;and their expressions of MAP2,GAP43 and GFAP detected by immunofluorescence staining.RESULTS Compared with the model group,the groups intervened with TUDCA,or BMSCs transplantation,or combination therapy shared improved hind limb function and spinal cord histomorphology(P＜0.05);increased fluorescence intensity of MAP2 and GAP43,and protein expressions of IL-10,Arg-1,p-PI3K and p-Akt(P＜0.05);decreased fluorescence intensity of GFAP and IL-6 protein expressions(P＜0.05);among which the combination therapy group took the lead(P＜0.05).CONCLUSION The combination therapy of TUDCA and BMSCs transplantation may restore the function of the rat model of SCI by reducing inflammatory reaction,alleviating secondary injury,and promoting axon and myelin regeneration via PI3K/Akt signaling pathway.

AIM:Hepatotoxicity of Brucea javani-ca picryl with broad-spectrum anticancer effect in nude mice based on hepatic drug metabolizing en-zyme CYP450 activity.METHODS:Fifty-six nude mice were randomly divided into blank group,Bru-cea javanica low-dose group(2 mg/kg),Brucea ja-vanica high-dose group(4 mg/kg),and cisplatin group(2 mg/kg),with 14 mice in each group.The blank group was injected with the same amount of normal saline every 3 days for 6 weeks.Calculate the mortality rate of nude mice in each group,ob-serve the general growth state of nude mice,re-cord the weight change of nude mice before and af-ter administration,weigh and record the liver weight after taking materials,and calculate the liv-er coefficient(liver weight/weight mass×100％),ob-serve and record the liver color and morphology.Hematoxylin-eosin(HE)staining was used to ob-serve the pathological changes of liver tissue.De-tection of alanine aminotransferase(ALT),aspar-tate aminotransferase(AST),lactate dehydrogenase(LDH),alkaline phosphatase(AKP)and albumin(ALB)levels in serum of nude mice by ELISA.Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of CYP2E1,CYP3A11,CYP2C19,CYP1A2,CYP2D6 and CYP2C9,which were key enzymes of drug metabolism in nude mice liver.RESULTS:Compared with the blank group,the mortality rate of nude mice in the low-dose Brucea javanica bitter alcohol group was 0,the growth state was good,the diet,movement,and mental state were normal,the weight change and liver coefficient ratio were consistent,the liver color was ruddy,the liver lobule morphology was complete under the microscope,the structure was clear,the liver cells were arranged regularly,and there was no inflammatory cell infiltration.There was no significant difference in the content of ALT,AST,LDH,AKP,and ALB.There was no significant difference in the mRNA and protein expression of CYP2E1,CYP3A11,CYP2C19,CYP1A2,CYP2D6,and CYP2C9(all P＞0.05).Compared with the blank group,the mortality rate of nude mice in the high-dose group of Brucea javanica bitter alcohol was 14.3％,the growth state was slightly poor,the diet,movement,and mental state were reduced,the weight growth was slow,the liver coefficient ratio was increased,the liver color was reddish brown,some liver lobule boundaries were unclear,a small number of liver cells were loosely arranged,the contents of ALT,AST,LDH,AKP,and ALB were signif-icantly increased,the mRNA levels of CYP2E1,CYP3A11,CYP2C19,CYP1A2,CYP2D6,and CYP2C9 were significantly reduced,and the protein expres-sions of CYP2E1,CYP3A11,CYP1A2,and CYP2D6 were significantly reduced(all P＜0.05 or P＜0.01),but there was no statistical difference in the mRNA and protein expression of CYP2C19,and the pro-tein expression of CYP2C9(P＞0.05).Compared with the blank group,the mortality rate of nude mice in the cisplatin group was 35.7％,the growth state was poor,the diet,action,and mental state were low,the weight gain was less,the liver coefficient ratio was significantly increased,the liver color was dark red,the liver sinusoids and central veins were congested,the hepatocytes were disordered,the nuclei were consolidated and contracted,and the arrangement was loose,the contents of ALT,AST,LDH,AKP,and ALB were significantly increased,and the mRNA and protein expressions of CYP2E1,CYP3A11,CYP2C19,CYP1A2,CYP2D6,and CYP2C9 were significantly reduced(all P＜0.05 or P＜0.01).CONCLUSION:The dose of Brucea javanica bitter alcohol is correlated with hepatotoxicity to nude mice.High doses of Brucea javanica bitter alcohol have hepatotoxicity to nude mice,which may be re-lated to reducing serum levels of ALT,AST,LDH,AKP,and ALB,inhibiting the expression of multiple subtypes of enzymes in the key enzyme CYP450 of liver drug metabolism,and then reducing the me-tabolism of toxic substances.

Objective To summarize the changing prevalence of carbapenem resistance in Enterobacterales based on the data of CHINET Antimicrobial Resistance Surveillance Program from 2015 to 2021 for improving antimicrobial treatment in clinical practice.Methods Antimicrobial susceptibility testing was performed using a commercial automated susceptibility testing system according to the unified CHINET protocol.The results were interpreted according to the breakpoints of the Clinical & Laboratory Standards Institute(CLSI)M100 31st ed in 2021.Results Over the seven-year period(2015-2021),the overall prevalence of carbapenem-resistant Enterobacterales(CRE)was 9.43％(62 342/661 235).The prevalence of CRE strains in Klebsiella pneumoniae,Citrobacter freundii,and Enterobacter cloacae was 22.38％,9.73％,and 8.47％,respectively.The prevalence of CRE strains in Escherichia coli was 1.99％.A few CRE strains were also identified in Salmonella and Shigella.The CRE strains were mainly isolated from respiratory specimens(44.23±2.80)％,followed by blood(20.88±3.40)％and urine(18.40±3.45)％.Intensive care units(ICUs)were the major source of the CRE strains(27.43±5.20)％.CRE strains were resistant to all the β-lactam antibiotics tested and most non-β-lactam antimicrobial agents.The CRE strains were relatively susceptible to tigecycline and polymyxins with low resistance rates.Conclusions The prevalence of CRE strains was increasing from 2015 to 2021.CRE strains were highly resistant to most of the antibacterial drugs used in clinical practice.Clinicians should prescribe antimicrobial agents rationally.Hospitals should strengthen antibiotic stewardship in key clinical settings such as ICUs,and take effective infection control measures to curb CRE outbreak and epidemic in hospitals.

Objective To characterize the changing species distribution and antibiotic resistance profiles of respiratory isolates in hospitals participating in the CHINET Antimicrobial Resistance Surveillance Program from 2015 to 2021.Methods Commercial automated antimicrobial susceptibility testing systems and disk diffusion method were used to test the susceptibility of respiratory bacterial isolates to antimicrobial agents following the standardized technical protocol established by the CHINET program.Results A total of 589 746 respiratory isolates were collected from 2015 to 2021.Overall,82.6％of the isolates were Gram-negative bacteria and 17.4％were Gram-positive bacteria.The bacterial isolates from outpatients and inpatients accounted for(6.0±0.9)％and(94.0±0.1)％,respectively.The top microorganisms were Klebsiella spp.,Acinetobacter spp.,Pseudomonas aeruginosa,Staphylococcus aureus,Haemophilus spp.,Stenotrophomonas maltophilia,Escherichia coli,and Streptococcus pneumoniae.Each microorganism was isolated from significantly more males than from females(P＜0.05).The overall prevalence of methicillin-resistant S.aureus(MRSA)was 39.9％.The prevalence of penicillin-resistant S.pneumoniae was 1.4％.The prevalence of extended-spectrum β-lactamase(ESBL)-producing E.coli and K.pneumoniae was 67.8％and 41.3％,respectively.The overall prevalence of carbapenem-resistant E.coli,K.pneumoniae,Enterobacter cloacae,Pseudomonas aeruginosa,and Acinetobacter baumannii was 3.7％,20.8％,9.4％,29.8％,and 73.3％,respectively.The prevalence of β-lactamase was 96.1％in Moraxella catarrhalis and 60.0％in Haemophilus influenzae.The H.influenzae isolates from children(＜18 years)showed significantly higher resistance rates to β-lactam antibiotics than the isolates from adults(P＜0.05).Conclusions Gram-negative bacteria are still predominant in respiratory isolates associated with serious antibiotic resistance.Antimicrobial resistance surveillance should be strengthened in clinical practice to support accurate etiological diagnosis and appropriate antimicrobial therapy based on antimicrobial susceptibility testing results.

In proton therapy,prompt gamma-ray imaging is considered as one of the most promising methods for assessing proton beam range.Prompt gamma-ray imaging detector evaluates the proton beam range based on the prompt gamma-ray distribution obtained by the prompt gamma-ray imaging system,which enables high-precision measurement of the proton beam range.Herein a proton beam range verification algorithm is designed for the newly developed prototype of the range verification detector(pixelated prompt gamma-ray imaging detector),which verifies the range estimation accuracy of the prototype for different phantoms and different energies of homogeneous media through Monte Carlo simulation.The results show that the accuracy of the proton beam range verification algorithm is within 0.5 mm of the safety margin error of the Bragg peak,and the measurement accuracy is significantly improved with the increase of the number of protons,indicating that the prototype algorithm is feasible for proton beam range verification.