Tujia ethnic group medicine Xuetong is derived from Kadsura heteroclita, the stem of which has the medicinal value for anti-rheumatoid arthritis, liver protection, anti-tumor, anti-oxidation effects, and has been widely used in Hunan and Guangdong in China. The selection of reliable and stable reference genes is the basis for subsequent molecular research on K. heteroclita. In this study, GAPDH, TUA, Actin, UBQ, EF-1α, 18S-rRNA, CYP, UBC, TUB, H2A, and RPL were selected as candidate reference genes in Kadsura heteroclita. The gene expression levels of the 11 candidate reference genes of K. heteroclita in its 6 different parts(stem-inside of the cambium, stem-outside of the cambium, fruit, flower, root, and leaf) and under different intervention conditions [drought stress, salt stress, and methyl jasmonate(MeJA) treatment] were detected by quantitative real-time polymerase chain reaction(qRT-PCR). The expression stability of the 11 candidate reference genes was comprehensively analyzed and evaluated by geNorm, NormFinder, ΔCT algorithm, and RefFinder software. The results showed that the expression of UBC and RPL was relatively stable in 6 different parts, and UBC and GAPDH genes were relatively stable under different intervention conditions. To verify the reliability of reference genes for K. heteroclita, this study further examined the relative expression levels of KhFPS, KhIDI, KhCAS, KhSQE, KhSQS, KhSQS-2, KhHMGS, KhHMGR, KhMVD, KhMVK, KhDXR, KhDXS, KhPMVK, and KhGGPS in different parts and under different intervention conditions, which might relate to the synthesis of the main component(Xuetongsu) of K. heteroclita. The results showed that with UBC and RPL or UBC and GAPDH as the reference genes, the expression trends of these 14 genes were basically consistent in different parts or under different intervention conditions for K. heteroclita. In conclusion, UBC can be used as a reference gene of K. heteroclita for its different parts and different intervention conditions, which lays a foundation for further research on the biosynthetic pathway of main components in K. heteroclita.
Real-Time Polymerase Chain Reaction/methods* ; Reference Standards ; Gene Expression Regulation, Plant ; Gene Expression Profiling ; Plant Proteins/metabolism* ; Drugs, Chinese Herbal

Objective To investigate the effect and mechanism of pomalidomide(POM)on airway inflammation and mucus hypersecretion in rats with chronic obstructive pulmonary disease(COPD).Methods Thirty-six SD rats were randomly divided into control group,model group and POM group,with 12 in each group,half male and half female.The COPD model was established by smoke exposure combined with Klebsiella pneumoniae infection in model group and POM group.The rats in POM group were treated with POM(0.5 mg/kg,once a day for 1 week).The lung function,lung tissue pathology,the proportion of inflammatory cells in bronchoalveolar lavage fluid(BALF)and the levels of serum inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 and IL-13 were observed and detected in each group.AB-PAS staining and immunohistochemistry were used to analyze the proliferation of goblet cells and the secretion of mucin(MUC)5AC and MUC5B in airway epithelium of rats.The expression levels of TNF-α receptor 1(TNFR1),IκB kinase(IKK),phosphorylated IKK(p-IKK)and P65 protein in lung tissue were detected by Western blotting.Results Compared with control group,model group showed significant decreased of tidal volume(TV),minute ventilation(MV),forced expiratory vital capacity(FVC),0.1s forced expiratory volume(FEV0.1)and 0.3 s forced expiratory volume(FEV0.3)(P<0.05),increased of the mean linear intercept(MLI)of the alveoli(P<0.01),decreased of the mean alveolar number(MAN)(P<0.01),increased of the proportion of neutrophils and lymphocytes in BALF sediment(P<0.05),and decreased of the proportion of macrophages in BALF sediment(P<0.01);increased of the levels of serum inflammatory factors TNF-α,IL-1β,IL-13 and IL-6(P<0.05),the proportion of goblet cells in airway epithelium(P<0.01),the secretion of MUC5AC and MUC5B in lung tissue(P<0.01),the content of TNFR1 and the ratio of p-IKK/IKK(P<0.01),the content of P65 in nucleus(P<0.01);and decreased of the content of P65 in cytoplasm(P<0.05).Compared with model group,after one week of POM treatment,POM group showed significant improved of the TV,MV,FVC,FEV0.1,FEV0.3,MLI and MAN of rats(P<0.05);decreased of the proportion of neutrophils and lymphocytes in BALF(P<0.05);increased of the proportion of macrophages(P<0.01);decreased of the levels of serum TNF-α,IL-1β,IL-6 and IL-13(P<0.05),the proportion of goblet cells in airway(P<0.01),the secretion of MUC5AC and MUC5B(P<0.01),and the expression of TNFR1,P-IKK and P65(nucleus)(P<0.05);and increased of the level of P65(cytoplasm)(P<0.01).Conclusions POM can improve airway inflammation and mucus hypersecretion in COPD rats,which may be achieved by inhibiting TNF-α/NF-κB signaling pathway.

A male infant,aged 6 days,was admitted to the hospital due to respiratory distress and systemic desquamative rash after birth.The infant presented with erythema and desquamative rash,respiratory failure,recurrent infections,chronic diarrhea,hypernatremic dehydration,and growth retardation.Comprehensive treatment,including anti-infection therapy,intravenous immunoglobulin administration,and skin care,resulted in improvement of the rash,but recurrent infections persisted.Second-generation sequencing revealed a homozygous mutation in the SPINK5 gene,consistent with the pathogenic variation of Netherton syndrome.The family opted for palliative care,and the infant died at the age of 2 months after discharge.This report documents a case of Netherton syndrome caused by the SPINK5 gene mutation in the neonatal period,and highlights multidisciplinary diagnosis and therapy for this condition.

Aim To design the pyrimidoindole deriva-tive N-butyl-9H-pyrimido[4,5-b]indole-2-carboxamide(BFPI)and synthesize it to investigate whether it in-hibits macrophage pyroptosis and foaming effects through the NLRP3/Caspase-1 pathway.Methods BFPI was synthesized using 2,4,6-triethoxycarbonyl-l,3,5-triazine and 2-aminoindole as starting materials and structurally characterized by 1H NMR,13C NMR,and ESI-MS.The in vitro cultured mouse monocyte macro-phage cell line RAW264.7 was divided into blank,model(PA)and therapeutic(BFPI)groups,and the cells in each group were treated with the corresponding culture medium for 24 h.The proliferative viability was detected by MTT assay,and the formation of intracel-lular lipid droplets was detected by oil red O staining,and NLRP3 was detected by Western-blot and RT-qPCR,caspase-1 and MCP-1 mRNA and protein ex-pression levels by Western blot and RT-qPCR.Results Compared with the blank group,the proliferation vi-ability of cells in the model group significantly de-creased and the formation of lipid droplets significantly increased;compared with the model group,the prolif-eration viability of cells in the treatment group signifi-cantly increased and the formation of lipid droplets sig-nificantly decreased,and the differences were statisti-cally significant(P＜0.01);compared with the blank group,the cellular NLRP3,caspase-1 and MCP-1 mR-NA and protein expression levels of cells in the model group significantly increased;compared with the model group,the expression levels of the above indexes of the cells in the treatment group significantly decreased,and the difference was statistically significant(P＜0.01).Conclusions BFPI contributes to delaying macrophage-derived foam cell formation during athero-genesis by inhibiting macrophage NLRP3,caspase-1,and MCP-1 expression and thereby promoting their pro-liferation and inhibiting lipid phagocytosis.

Objective To explore the causal relationship between ischemic stroke and different types of epilepsy so as to provide genetic evidence for the risk of epilepsy after ischemic stroke.Methods Based on the summary data of genome-wide association study,Mendelian randomization (MR ) analysis was performed with MR-Egger regression,weighted median and inverse-variance weighting.Sensitivity analysis was conducted by using MR-Egger regression test,MR-PRESSO test and leave-one-out method.Results Two-sample MR analysis showed that there was a causal relationship between ischemic stroke and the risk of epilepsy (OR=1.10,P=0.036)and generalized epilepsy (OR=1.25,P=0.001 ),but no such relationship was observed with the risk of focal epilepsy (OR=1.06,P=0.360).In addition,diastolic blood pressure (OR=1.12,P=0.002)and BMI (OR=1.08,P=0.015)also had a causal relationship with the risk of epilepsy. After adjusting confounding factors,multivariate MR analysis revealed that only ischemic stroke (OR=1.18,P=3.770×10-6)and epilepsy risk had a causal relationship.Conclusion There is a causal relationship between ischemic stroke and the risk of generalized epilepsy,which can be used to stratify the risk of post-stroke epilepsy.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

The male patient,one day old,was admitted to the hospital due to hypoglycemia accompanied by apnea appearing six hours after birth.The patient had transient hypoglycemia early after birth,and acute heart failure suddenly occurred on the eighth day after birth.Laboratory tests showed significantly reduced levels of adrenocorticotropic hormone and cortisol,and pituitary magnetic resonance imaging was normal.Genetic testing results showed that the patient had probably pathogenic compound heterozygous mutations of the TBX19 gene(c.917-2A＞G+c.608C＞T),inherited respectively from the parents.The patient was conclusively diagnosed with congenital isolated adrenocorticotropic hormone deficiency caused by mutation of the TBX19 gene.Upon initiating hydrocortisone replacement therapy,cardiac function rapidly returned to normal.After being discharged,the patient continued with the hydrocortisone replacement therapy.By the 18-month follow-up,the patient was growing and developing well.In neonates,unexplained acute heart failure requires caution for possible endocrine hereditary metabolic diseases,and timely cortisol testing and genetic testing should be conducted.[Chinese Journal of Contemporary Pediatrics,2024,26(3):321-324,V]

Objective To explore the effect of gastrodin(GAS)on the polarization of M2 microglia under oxygen and glucose deprivation(OGD)and the effect of p38 MAPK inhibition on the polarization status of microglia.Methods BV2 microglia was divided into the control group(Ctrl),p38 MAPK inhibitor SB203580 group(Ⅰ),OGD group(OGD),OGD+I group,gastrodin treatment group(G+OGD),and G+OGD+Ⅰ group.The expressions of p38 MAPK,phosphorylated p38 MAPK(p-p38 MAPK),M2 microglia marker arginase-1(Arg-1)and chitinase like protein 1/2(YM1/2)were detected by immunofluorescent staining and Western blotting.Results Immunofluorescent staining results showed that the pretreatment with gastrodin reduced the fluorescence expression of p-p38 MAPK in OGD induced BV2 microglia,enhanced the fluorescence expressions of Arg-1 and YM1/2.After pretreatment with SB203580,the fluorescence expressions of p-p38 MAPK further decreased,the fluorescence expressions of Arg-1 and YM1/2 further increased,both were significantly different from the G+OGD group(n=3,P＜0.05).The fluorescence expressions of p38 MAPK in each group was not statistically significant(n=3,P＞0.05).Western blotting results showed that the protein expressions of p-p38 MAPK,Arg-1 and YM1/2 in the OGD group enhanced,compared with that in the control group(n=3,P＜0.05).After the pretreatment with gastrodin,the protein expression of p-p38 MAPK was reduced significantly,the protein expressions of Arg-1 and YM1/2 was enhanced obviously,both were significantly different from the OGD group(n=3,P＜0.05).After pretreatment with SB203580,the protein expressions of p-p38 MAPK was further reduced,the protein expression of Arg-1 and YM1/2 were further enhanced,both were significantly different from the G+OGD group(n=3,P＜0.05).In addition,there was no significant change in p38 MAPK protein expression among groups(n=3,P＞0.05).Conclusion Gastrodin inhibits p38 MAPK signaling activation to promote BV2 microglial polarization towards the M2 phenotype,reducing inflammatory responses and exerting a protective effect.

Objective To investigate the effects of Simo decoction on duodenal microinflammation and NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate-specific proteinase-1(Caspase-1)/gasdermin D(GSDMD)signaling pathway in rats with functional dyspepsia(FD).Methods The FD model was established by multifactorial method.SD rats were randomly divided into normal group,model group(FD model),positive control group(gavage administration of 0.305 mg·kg-1 mosapride injection)and experimental-H,-M,-L groups(gavage administration of 5.62,2.81,1.40 g·kg-1 Simo decoction).Small intestinal advancement rate and gastric emptying rate was determined;the levels of interleukin(IL)-1 β and IL-18 in serum were determined by enzyme linked immunosorbent assay(ELISA);the protein expression of NLRP3 and GSDMD in duodenal tissue was detected by Western blotting.Results The gastric emptying rates of normal,model,positive control and experimental-H,-M,-Lgroupswere(58.34±5.72)％,(29.16±8.37)％,(48.77±6.10)％,(48.35±6.04)％,(48.20±3.49)％and(39.24±4.20)％;the small intestinal propulsion rates were(82.01±7.55)％,(41.95±9.53)％,(64.61±10.18)％,(75.04±9.76)％,(60.58±7.13)％and(45.89±7.40)％;serum IL-1 β expression were(12.86±0.88),(43.73±4.60),(18.84±0.86),(24.61±1.57),(19.14±0.77)and(29.04±0.72)pg·mL-1;IL-18 expressions were(95.00±3.74),(170.60±8.78),(108.50±3.05),(118.90±3.45),(99.90±8.70)and(141.00±3.71)pg·mL-1;the relative expression levels of NLRP3 proteins were 0.32±0.02,0.84±0.05,0.42±0.03,0.48±0.02,0.61±0.04 and 0.62±0.05;the relative expression levels of GSDMD proteins were 0.34±0.05,0.93±0.06,0.35±0.03,0.52±0.02,0.53±0.06 and 0.55±0.05,respectively.Compared with the normal group,the above indexes in the model group have statistical significance;compared with the model group,the above indexes in the experimental-H group and the positive control group also have statistical significance(P＜0.01 or P＜0.05).Conclusion Simo decoction can effectively improve the general condition and duodenal microinflammation in FD rats,and the mechanism may be related to the inhibition of duodenal NLRP3/Caspase-1/GSDMD signaling pathway.