ObjectiveTo evaluate pharmacological effects of Shengmai injection（SMI）on cerebral ischemia and study its neuroprotective mechanism. MethodsMale specific pathogen-free （SPF） Sprague-Dawley （SD） rats were randomly divided into a sham group， a model group， a low-dose SMI group（3 mL·kg^-1）， a middle-dose SMI group（6 mL·kg^-1）， a high-dose SMI group（12 mL·kg^-1）， and a Ginaton group（4 mL·kg^-1）according to the random number table method， with 12 rats in each group. The rat model of cerebral ischemia-reperfusion（MCAO/R）was prepared via the suture method. The administration groups were intraperitoneally injected with corresponding concentrations of SMI or Ginaton injection after reperfusion， which was conducted for 3 consecutive days. The sham group and model group were administered the equivalent volume of physiological saline. The pharmacological effects of SMI on brain injury in MCAO/R rats were evaluated by neurological function scores， cerebral infarction area， hematoxylin-eosin （HE） staining， Nissl staining， terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） staining， and Western blot. The dominant link and key protein of SMI treating cerebral injury were explored using proteomic analysis. The related mechanisms of SMI were further validated using enzyme-linked immunosorbent assay （ELISA）， Western blot， and chloride ion fluorescence probe with oxygen-glucose deprivation/reoxygenation（OGD/R）-treated PC12 cells and MCAO/R rats. ResultsCompared with the sham group， the model group showed significantly increased neurological function scores， cerebral infarction area， neuronal apoptosis rate， and expression levels of apoptosis related proteins （P<0.05， P<0.01）and significantly decreased density of Nissl bodies and neurons（P<0.01）. Compared with the model group， the SMI groups exhibited significantly decreased neurological function scores， cerebral infarction area， neuronal apoptosis rate， and expression levels of apoptosis related proteins （P<0.05， P<0.01）and significantly increased density of Nissl bodies and neurons （P<0.05）. The proteomic analysis results showed that oxidative stress and inflammatory response were important processes of SMI intervening in MCAO/R injury， and the chloride intracellular channel protein 1 （CLIC1） was one of key proteins in its action network. The levels of representative indicators of oxidative stress and inflammatory response in the MCAO/R rats of the SMI groups were significantly reduced， compared with those in the model group（P<0.05， P<0.01）， and the expression levels of CLIC1 and downstream NOD-like receptor protein 3 （NLRP3） decreased （P<0.01）. In addition， the experimental results based on the OGD/R PC12 cells showed that SMI significantly increased the cell survival rate（P<0.01） and significantly decreased the intracellular chloride ion concentration（P<0.05）. ConclusionSMI has neuroprotective effects. Oxidative stress and inflammatory response are key processes of SMI intervening in MCAO/R injury. The potential mechanism is closely related to the regulation of CLIC1.

ObjectiveBased on the integrated strategy of "empirical prescriptions in ancient books-medical cases by prestigious doctors-computational analysis"， this study aims to explore and analyze the prescriptions and medical cases for treating tremors in traditional Chinese medicine （TCM）， predict their efficacy， and obtain the core prescriptions for treating tremors in TCM， providing references for clinical application and new drug development. MethodThe Chinese Medicine Prescription Database and the China National Knowledge Infrastructure （CNKI） were searched for relevant prescriptions and medical cases for treating tremors in TCM to establish a database of prescriptions for tremors. The Traditional Chinese Medicine Inheritance Computer System （V3.0） was used to analyze and explore the medication rules including drug frequency， properties， flavor， meridian tropism， and pharmacological effects， as well as core drugs and formula associations. A multi-target drug efficacy prediction platform based on network robustness was used to evaluate the predicted efficacy of the core prescriptions obtained. Based on the integration of ancient prescriptions， prestigious doctors' medical cases， and network analysis results， the priority level of the developed prescriptions was determined through comprehensive evaluation. ResultA total of 81 ancient prescriptions were screened， involving 246 drugs， and 171 prescriptions were screened from prestigious doctors' medical cases， involving 278 drugs. The frequently used TCM drugs were mostly warm in nature and sweet in flavor， mainly acting on the liver， spleen， and kidney meridians. In terms of efficacy， they were mainly effective in tonifying deficiency， soothing liver and extinguishing wind， activating blood and resolving blood stasis， clearing heat， and resolving exterior. Through association rules and K-means clustering， the core prescriptions were composed of high-frequency drugs such as Glycyrrhizae Radix et Rhizoma， Ginseng Radix et Rhizoma， Astragali Radix， Atractylodis Macrocephalae Rhizoma， Angelicae Sinensis Radix， Poria， Gastrodiae Rhizoma， and Uncariae Ramulus Cum Uncis. Drug combinations mainly focused on tonifying Qi and nourishing blood， with the additional functions of calming wind and dredging collaterals. Clustering analysis of core prescriptions from ancient prescriptions and prestigious doctors' medical cases， as well as multi-target drug efficacy prediction， showed that Combination 1 had the highest disturbance score on the disease network. Furthermore， comparative analysis revealed consistent results with both the analysis of ancient prescriptions and prestigious doctors' medical cases， indicating its optimal development potential based on theoretical inheritance and empirical practice. In comparison， Combinations 3， 2， and 4 were less utilized in contemporary clinical practice， with lower rankings in network disturbance scores， suggesting that their development value still warranted further exploration. ConclusionTCM clinical treatment of tremors emphasizes the regulation of the liver， spleen， and kidney. In line of syndrome differentiation， drugs potent in soothing liver， extinguishing wind， activating blood， and resolving blood stasis are added based on deficiency-tonifying drugs. The core prescriptions based on Glycyrrhizae Radix et Rhizoma， Angelicae Sinensis Radix， Paeoniae Radix Alba， Astragali Radix， Poria， and Atractylodis Macrocephalae Rhizoma （combination 1） have the highest potential development value. The integrated strategy "empirical prescriptions in ancient books-medical cases by prestigious doctors-computational analysis" can be used for the screening of candidate prescriptions for new TCM drugs.

BACKGROUND:Adjustable piezoelectric effect can promote tissue regeneration and repair.Piezoelectric materials are widely used in weight-bearing tissue engineering. OBJECTIVE:To prepare a piezoelectric film material that can promote bone regeneration,and to explore its structural characterization,electrical output performance,biocompatibility,and effect of electrical output on osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. METHODS:Using poly-3-hydroxybutyrateco/4-hydroxybutyrate(P34HB)as raw material,barium calcium stannate titanate powder(Ba0.94Ca0.06Sn0.08Ti0.92O3,BCST)was added according to mass ratios of 0%,5%,10%,15%,and 20%.Dichloromethane was added to solve P34HB,and the thickness of 150-200 μm BCST/P34HB piezoelectric film was prepared by vacuum drying method.After polarization in the oil bath,the surface morphology,crystal phase composition,piezoelectric coefficient and open circuit voltage were tested.The effect of BCST/P34HB electrical output at 110 Hz and 0.25 N force on the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells was tested. RESULTS AND CONCLUSION:(1)Scanning electron microscopy,X-ray diffraction,water contact angle,piezoelectric coefficient and electrical output performance tests showed that when the mass ratio of BCST increased to 20%,the BCST/P34HB piezoelectric film had good piezoelectric properties(d33=5.9 pC/N)and electrical output performance(180 mV),which was closer to the suitable range of 500 mV for electrical stimulation.(2)Live and dead staining showed that on the first day of co-culture,15%group and 20%group showed less red fluorescence.On the 5th day of culture,the number of green fluorescence in each group was significantly higher than that on the first day,and the red fluorescence was not observed in the 10%,15%and 20%groups,and only a small amount of red fluorescence was observed in the 0%and 5%groups.(3)On the 1st,3rd and 5th days of co-culture with rabbit bone marrow mesenchymal stem cells,Almar blue staining exhibited that the number of cells in each group showed an increasing trend with the increase of time.On the 5th day of culture,the number of cells in the 20%group was significantly more than that in the 0%group(P<0.05).(4)On day 10 of osteogenic induction,alkaline phosphatase staining results showed that the positive rate of the 20%group was significantly higher than that of the 0%group(P=0.000 1).On day 21,alizarin red staining and quantitative analysis of calcium nodules showed a similar trend to alkaline phosphatase staining.Compared with the 0%group,the 15%group and 20%group showed significant differences(P<0.01,P<0.000 1).(5)The results showed that 20%BCST/P34HB films had good piezoelectric properties,electrical output properties,biocompatibility and the ability of promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

ObjectiveTo explore the mechanism of Naoxintong capsules' intervention in cerebral ischemia-reperfusion by building a mouse cerebral ischemia-reperfusion model based on short-chain fatty acids. MethodC57BL/6J male mice were randomly divided into the sham group， model group， Naoxintong group （158.9 mg∙kg^-1）， and Ginaton group （12.1 mg∙kg^-1） according to the random number table method. The model of cerebral ischemia-reperfusion （MCAO/R） was prepared via the filament occlusion method. The effect of Naoxintong capsules on brain injury in MCAO/R mice was evaluated by the neuroethological score， cerebral infarction area determination， Nissl staining， and immunofluorescence staining. Hematoxylin-eosin （HE） staining and Western blot were employed to evaluate the effect of Naoxintong capsules on the intestinal barrier in MCAO/R mice. The content of short-chain fatty acids in mouse feces was detected by LC-MS/MS. ResultCompared to the sham group， the model group exhibited significant increases in the cerebral infarction area， neuroethological score， and cell apoptosis rate （P<0.01）， with a notable decrease in the number of Nissl bodies （P<0.01）. The protein expression levels of Claudin-1 and Occludin were significantly reduced （P<0.05）. Compared with the model group， the intervention of Naoxintong capsules significantly decreased the cerebral infarction area （P<0.05） and improved the neuroethological score （P<0.01） and cell apoptosis rate （P<0.01）， with the number of Nissl bodies （P<0.01） and expression levels of Claudin-1 and Occludin proteins （P<0.01） increased. LC-MS/MS results showed that compared to the sham group， the model group featured a significantly reduced content of acetic acid， propionic acid， and butyric acid in feces （P<0.01）， while valeric acid， isovaleric acid， and isobutyric acid levels were increased （P<0.01）. The intervention of Naoxintong capsules notably lowered the content of valeric acid， isovaleric acid， and isobutyric acid （P<0.01）. ConclusionNaoxintong capsules can improve brain and intestinal barrier damage and play a protective role in cerebral ischemia-reperfusion by regulating the content of short-chain fatty acids.

Objective:To establish an immortalized human keloid fibroblasts(KFbs) cell line and identify its characteristics and functions.Methods:The specimen was obtained from a 32-year-old female patient who underwent surgical resection of an earlobe keloid at Peking University Third Hospital in November 2019. The keloid tissue obtained was removed from the subcutaneous fat and epidermis. It was then separated and cultured using the tissue sticking method to obtain primary KFbs, which were passaged using the trypsin digestion method. After the primary KFbs were infected with an SV40 lentivirus, purified by puromycin, and passaged, a human KFbs cell line was established. Chromosomal karyotype analysis, short tandem repeat (STR) profiling, and gender gene detection were conducted to identify the primary KFbs and the cell line. The CCK-8 method was used to assess the proliferation ability of the cells. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression levels of specific genes (PGK1, ENO1, LDHA, GLUT1, TGF-β1, COL1, COL3, FN). The comparative analysis of relevant data between primary KFbs and the cell line was conducted using t-test, and P<0.05 indicated statistical significance. Results:The morphology of both the primary KFbs and the cell line was typically spindle-shaped. The cell line morphology was basically similar to that of the primary KFbs, which were continuously cultured and passaged for 20 generations. The gender gene(Amelogenin) detection showed both were females. The chromosome karyotyping of the primary KFbs and cell line was satisfactory, maintaining the fundamental characteristics of normal cells without undergoing malignant transformation. The STR identification results showed that no multiple alleles were found in the cell line, indicating a normal cell genotype. Furthermore, the cell line did not match any entries in known cell databases. After 24, 48, and 72 hours of culture, the proliferation ability of the cell line increased by 76.1%, 125.8%, and 60.3% compared to primary KFbs. The proliferation rates of the cell line were significantly faster than those of primary KFbs ( P<0.05). The mRNA and protein expression levels of the aforementioned genes in the cell line showed no significant changes compared to the primary KFbs ( P>0.05). Conclusion:An immortalized human KFbs cell line was successfully established, showing no significant changes in morphology, characterization, and function, while exhibiting a faster proliferation rate compared to that of primary KFbs.

Objective To investigate the food allergy situation of children in Changping District of Beijing,and to explore the influence of allergic family history,gender and mode of delivery on food allergy in children,the distribution of food allergy in different age groups,the types of food allergy that are easy to cause in this area,and the comorbidities of food allergy.Methods A total of 515 children aged 0 to 14 years who were admitted to the general pediatric outpatient and emergency department and inpatient of Beijing Changping Hospital from April to November 2023.Using immunoblotting to detect specific immunoglobulin E in the serum of pediatric patients,and using SPSS 26.0 statistical software to perform binomial tests on gender and delivery mode non parameters;Using custom Excel functions to statistically analyze the family history of allergies,the number of people in different age groups,and the frequency of allergic foods in each group;Use a self-made mini program to statistically analyze the combination of comorbid allergic diseases.Results ① The number of male children with food allergies(306 cases)was higher than that of female children(209 cases),and the difference was statistically significant(P＜0.05).(2)There were 109 cases of pediatric patients with parents who had no history of allergic diseases,accounting for 21.17％of the total cases;There were a total of 406 cases where at least one parent had a family history of allergies,accounting for 78.83％of the total cases.Among them,228 cases(44.27％)had one parent with a history of allergies,and 178 cases(34.56％)had both parents with a history of allergies.③ Among 515 children with positive food allergens,there were 10 cases(1.94％)in infancy,37 cases(7.19％)in early childhood,235 cases(45.63％)in preschool,192 cases(37.28％)in school age,and 41 cases(7.96％)in adolescence.The highest positivity rate for food allergens is in milk(444 times),followed by egg white(70 times),cashew nuts(57 times),crab(37 times),beef(26 times),mango(24 times),shrimp(21 times),pineapple(6 times),and shellfish(1 time).Milk protein is the most common allergen in all age groups.(4)Among 515 children with food allergies,399 cases were single food allergies,accounting for 77.47％;116 cases of multiple food allergies(2 or more types of food allergies),accounting for 22.53％.⑤ The most common comorbidity of food allergies is food allergy related gastrointestinal diseases combined with allergic rhinitis,with a total of 267 cases;Secondly,there were 192 cases of allergic rhinitis combined with chronic cough,and 124 cases of food allergy related gastrointestinal diseases combined with chronic cough.Conclusion Milk is the main allergen of food allergy in people of age 14 and under,and gastrointestinal symptoms are the most common in children with food allergy.

Objective:To study the effect of tertiary lymphoid structures(TLS)on the pathological response and prognosis of patients with non-small cell lung cancer(NSCLC)receiving neoadjuvant therapy.Methods:We retrospectively collected the data of 132 patients with NSCLC who underwent neoadjuvant therapy and surgery at Tianjin Chest Hospital between January 2019 and December 2023,including 40 in the neoadjuvant chemotherapy(NC)group and 92 in the NC plus immunotherapy(NCI)group.The percentage of residual viable tumor(RVT)and tumor infiltrating lymphocyte(TIL)counts were evaluated by hematoxylin and eosin(H&E)staining,while TLS number and matur-ity were assessed by H&E and immunohistochemical staining.The differences in TLS number and maturity and effects on patient pathologic-al response and prognosis were compared between groups.Results:TIL count,total TLS number,pathological complete response and major pathological response rates were significantly higher in the NCI versus NC group(P<0.001).Moreover,a multivariate Logistic analysis sho-wed that TLS number and maturity and TIL count affected pathological response in the NCI group(P<0.05).A multiple linear regression ana-lysis indicated that a low TIL count was a risk factor for a high RVT in the NC group,while a low number of mature TLS,low TIL count,and N stage were independent risk factors for a high RVT in the NCI group(all P<0.05).In the NCI group,a multivariate Cox regression analysis showed that a low number of mature TLS(P=0.001)and low TIL count(P=0.009)were independent predictors of disease-free survival(DFS),while a survival analysis showed that patients in the NCI group with high(vs.low)numbers of mature TLS and a high(vs.low)TIL count had significantly longer DFS(all P<0.001).Conclusions:A low number of mature TLS and low TIL count were associated with an adverse patholo-gical response and short DFS in patients with NSCLC.Thus,TLS maturity and TIL count can predict the pathological response and prognosis of patients with NSCLC treated with NCI.

Objective:To establish an immortalized human keloid fibroblasts(KFbs) cell line and identify its characteristics and functions.Methods:The specimen was obtained from a 32-year-old female patient who underwent surgical resection of an earlobe keloid at Peking University Third Hospital in November 2019. The keloid tissue obtained was removed from the subcutaneous fat and epidermis. It was then separated and cultured using the tissue sticking method to obtain primary KFbs, which were passaged using the trypsin digestion method. After the primary KFbs were infected with an SV40 lentivirus, purified by puromycin, and passaged, a human KFbs cell line was established. Chromosomal karyotype analysis, short tandem repeat (STR) profiling, and gender gene detection were conducted to identify the primary KFbs and the cell line. The CCK-8 method was used to assess the proliferation ability of the cells. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression levels of specific genes (PGK1, ENO1, LDHA, GLUT1, TGF-β1, COL1, COL3, FN). The comparative analysis of relevant data between primary KFbs and the cell line was conducted using t-test, and P<0.05 indicated statistical significance. Results:The morphology of both the primary KFbs and the cell line was typically spindle-shaped. The cell line morphology was basically similar to that of the primary KFbs, which were continuously cultured and passaged for 20 generations. The gender gene(Amelogenin) detection showed both were females. The chromosome karyotyping of the primary KFbs and cell line was satisfactory, maintaining the fundamental characteristics of normal cells without undergoing malignant transformation. The STR identification results showed that no multiple alleles were found in the cell line, indicating a normal cell genotype. Furthermore, the cell line did not match any entries in known cell databases. After 24, 48, and 72 hours of culture, the proliferation ability of the cell line increased by 76.1%, 125.8%, and 60.3% compared to primary KFbs. The proliferation rates of the cell line were significantly faster than those of primary KFbs ( P<0.05). The mRNA and protein expression levels of the aforementioned genes in the cell line showed no significant changes compared to the primary KFbs ( P>0.05). Conclusion:An immortalized human KFbs cell line was successfully established, showing no significant changes in morphology, characterization, and function, while exhibiting a faster proliferation rate compared to that of primary KFbs.

Objective To study the causes of two Chinese families with Branchio-oto syndrome.Methods The clinical data of two families were collected,and the pathogenic genes and variants of Branchio-oto syndrome were screened and verified by whole exome sequencing and Sanger sequencing.Results Two proband patients were diagnosed with Branchio-oto syndrome.Proband 1 presented with preauricular and anterior cervical fistulas,as well as congenital severe sensorineural hearing loss.On the other hand,proband 2 displayed a preauricular fistula and an anterior cervical cyst.At the age of 5,progressive deterioration of binaural hearing was observed,leadingtothe cur-rent diagnosis of severe mixed deafness.Genetic analysis showed that proband 1 and 2 carried nonsense variants of EYA1 gene:NM_000503.6:c.1408G＞T(p.Glu470Ter),and c.889C＞T(p.Arg297Ter).According to the guide-lines of the American College of Medical Genetics and Genomics(ACMG),the above variants were rated as patho-genic variants.After reviewing the literature,the c.1408G＞T variant had not been previously reported,and the c.889C＞T is a known variant.Conclusion The variants c.1408G＞T(p.Glu470Ter)and c.889C＞T(p.Arg297Ter)of EYA1 gene are the cause of these two families with Branchio-oto syndrome.The first report of c.1408G＞T broadens the mutational spectrum of EYA1 gene and provids a clinical reference for the diagnosis of Branchio-oto syndrome.

Objective To investigate ecological isolation between Oncomelania hupensis snail populations in hilly regions and marshland and lake regions in Yuanjiang valley, Changde City, Hunan Province, and to unravel its underlying mechanisms. Methods Taoyuan County, Shimen County, Linli County and Lixian County in Changde City were selected as snail sampling sites in hilly regions, and Lixian County, Jinshi City, West Lake Administration District, Hanshou County and Dingcheng District were selected as snail sampling sites in marshland and lake areas. Cytochrome C oxidase 1 (cox 1) gene was amplified in snail samples and sequenced. The genetic sequences of O. hupensis snails were aligned using the software MEGA 11, and the haplotypes of O. hupensis snails were determined using the software DNASP 5.10.01. The phylogenetic tree was generated using Bayesian inference with the software MrBayes 3.2, and analysis of molecular variance (AMOVA) was performed to analyze the source of genetic divergence and estimate the genetic divergence index (F_ST) among snail populations with the software Arlequin 3.5.2.2. The genetic barrier among 11 O. hupensis snail populations was estimated using the Monmonier algorithm of adegenet toolkit in R package. The settings with “land in winter and water in summer” in the Yuanjian River section were divided into two categories according to the upstream and downstream, and the areas with “land in winter and water in summer” in the upstream and downstream were transformed into raster data, and then loaded into the software Fragstats 4 for analysis of landscape indicators. The trends in changes of digital elevation were extracted from the Yuanjiang River section based on the digital elevation model, and made three-dimensional visualization using the R package. Results The mitochondrial cox 1 gene were amplified in 165 O. hupensis snais from 11 sampling sites and sequenced, and a total of 152 valid gene sequences were obtained, with 46 haplotypes or 9 populations determined. No haplotype was shared in snails between Taoyuan County and Dingcheng District and Hanshou County along the downstream of the Yuanjiang River. The total area of settings with “land in winter and water in summer” was 617.66 hm² in the upsteram of the Yuanjiang River, which consisted of 473 patches, with each patch measuring 1.31 hm², the largest area index of 0.735 2, the landscape division index of 0.999 9, and the landscape shape index of 45.293 7. The total area of settings with “land in winter and water in summer” was 9 956.92 hm² in the downstream of the Yuanjiang River, which consisted of 771 patches, with each patch measuring 12.91 hm², the largest area index of 97.839 9, the landscape division index of 0.042 7, and the landscape shape index of 7.249 6. The area of settings with “land in winter and water in summer” was much larger in the downstream than that in the upstream of the Yuanjiang River, and the stronger landscape connectivity and non-remarkable alteration of riverbed elevation provided suitable habitats for snail breeding. Conclusion The hydrological and environmental characteristics of the upstream of the Yuanjiang River restrain the breeding and spread of O. hupensis, resulting in ecological isolation between Oncomelania hupensis in Taoyuan County and those in the downstream of Yuanjiang River.