ObjectiveThis paper aims to investigate the effects and mechanism of Mimenghua prescription in modulating the mitogen-activated protein kinase kinase （MEK）/rat sarcoma viral oncogene homolog （Ras）/rapidly accelerated fibrosarcoma kinase （Raf）/extracellular signal-regulated kinase （ERK） signaling pathway to inhibit inflammatory responses in a dry eye animal model. MethodsA total of 60 C57BL/6J mice （eight weeks old， half male and half female） were used in the experiment. Ten mice were randomly selected as the blank control group， while the remaining 50 were exposed to a controlled dry system and received instillation of 0.2% benzalkonium chloride （BAC） into the eyes for four weeks to establish a dry eye mouse model. After successful modeling， the mice were randomly divided into five groups： Model group， sodium hyaluronate group， and Mimenghua prescription groups with low dose （4.83 g·kg^-1）， medium dose （9.67 g·kg^-1）， and high dose （19.34 g·kg^-1）. The mice in the model group received an equal volume of normal saline via gavage for four weeks. The mice in the sodium hyaluronate group received instillation of sodium hyaluronate eye drops twice daily for 14 consecutive days. The tear secretion volume， tear film break-up time （TBUT）， and corneal fluorescein staining were evaluated once every two weeks. After four weeks of administration， mice were euthanized， and their lacrimal gland tissues and corneas were harvested. Hematoxylin-eosin （HE） staining was used to assess histopathological morphology. Western blot was performed to detect the protein expression levels of MEK， Ras， Raf， and ERK. Enzyme-linked immunosorbent assay （ELISA） was used to measure the contents and expressions of MEK， Ras， Raf， ERK， and interleukin （IL）-1β in lacrimal gland and corneal tissues of the mice in each group. Quantitative real-time polymerase chain reaction （Real-time PCR） was employed to determine mRNA expression levels of MEK， Ras， Raf， and ERK. ResultsThe Mimenghua prescription groups and the sodium hyaluronate group exhibited significantly increased tear secretion volume （P<0.05） and prolonged TBUT （P<0.05） after treatment. Ocular surface damage of mice was visibly recovered. Western blot results indicated that protein expression levels of MEK， Ras， Raf， and ERK in the lacrimal gland and corneal tissues were significantly downregulated in the sodium hyaluronate group and Mimenghua prescription group with high dose （P<0.05）. ELISA results showed that IL-1β levels were highest in the model group but significantly reduced in the sodium hyaluronate group and Mimenghua prescription groups （P<0.05）. Both ELISA and Real-time PCR results demonstrated that the expression levels of MEK， Ras， Raf， and ERK in the lacrimal glands and corneal tissues were significantly elevated in the model group （P<0.05）， but markedly downregulated in the sodium hyaluronate group and Mimenghua prescription groups （P<0.05）， suggesting that Mimenghua prescription can decrease the expressions of MEK， Ras， Raf， and ERK in the lacrimal glands and corneal tissues. ConclusionMimenghua prescription can reduce inflammatory responses， increase tear secretion， prolong TBUT， and promote corneal recovery by inhibiting the MEK， Ras， Raf， and ERK signaling pathways in lacrimal gland and corneal tissues.

ObjectiveThis paper aims to observe the syndrome improvement and negative antigen conversion rate of Qingxuan Daozhi formula in the treatment of influenza in children （heat accumulation in the lung and stomach syndrome）. MethodsThrough a multi-center randomized controlled methodology design，confirmed influenza cases were collected from October 2022 to April 2023 in the pediatrics department of eight hospitals，such as Dongfang Hospital of Beijing University of Chinese Medicine. A total of 180 children with influenza and heat accumulation in the lung and stomach syndrome conforming to the standard were recruited through the clinic. The sick children meeting the inclusion criteria were randomly divided into groups by a block-randomized method. The children in the experimental group were treated with Qingxuan Daozhi formula for five days，and those in the control group were treated with Oseltamivir Phosphate Granules for five days. The primary efficacy indicator was the negative conversion rate of influenza antigen detection. Secondary efficacy indicators were the Canadian acute respiratory illness and flu scale （CARIFS） and the incidence of complications，severe cases， and critical cases. Follow-up observation was conducted on the day of enrollment，48 hours after medication，72 hours after medication， and （6+1） d after medication. ResultsOne hundred and eighty participants were randomly assigned to the experimental group （90 cases） or the control group （90 cases）. All participants were followed up during the study. Comparison of influenza antigen detection results in the primary efficacy indicators showed that the average time of negative influenza antigen conversion in the experimental group was （5.29±1.25） d，and that in the control group was （5.40±1.68） d，without a statistically significant difference. After five days of intervention，52 cases in the experimental group and 51 cases in the control group converted to negative，without a statistically significant difference. CARIFS score results in the secondary efficacy indicators showed that during 72 hours after intervention，there were statistically significant differences between the experimental group and the control group in three dimensions， including headache，muscle soreness， and the need for extra care （P<0.05）. On the （6+1） days after the intervention，the differences in both the experimental group and the control group were statistically significant in 10 dimensions， including sore throat，bad sleep，uncomfortable feeling，poor spirit and fatigue，crying more than usual，the need for extra care，symptom，function，influence on parents，and total score （P<0.05）. The comparison results within the group in the dimensional scores of symptom， function， and influence on parents，as well as the CARIFS total score showed that with the delay of follow-up time，scores of both groups decreased significantly，with a statistically significant difference （P<0.01）. Inter-group comparison results showed that the mean score of the experimental group was higher than that of the control group at the time of enrollment. With the progress of intervention，the score of the experimental group was significantly decreased compared with that of the control group. At the end of follow-up，the mean score of the experimental group was lower than that of the control group，with no statistically significant difference. In terms of the incidence of complications，severe cases， and critical cases， there were no complications，severe cases， and critical cases in the two groups，without a statistically significant difference. ConclusionThe symptom improvement effect and negative antigen conversion rate of Qingxuan Daozhi formula in the treatment of influenza in children （heat accumulation in the lung and stomach syndrome） are not inferior to Oseltamivir Phosphate granules， and children's acceptance is better. It can be more widely used in clinical treatment of influenza in children （heat accumulation in the lung and stomach syndrome）.

Background: Adrenocortical hormones, particularly 11-oxygenated androgens, are pivotal in female reproductive health and fertility. Standardized detection kits and population-specific reference intervals are lacking in China, hindering related clinical applications. Methods: A HPLC-tandem mass spectrometry (HPLC-MS/MS) pipeline was developed, rigorously validated, and applied to simultaneously quantify corticosterone, cortisone, cortisol, 18-OH cortisol, androstenedione (A4), 11β-hydroxyandrostenedione (11-OH A4), dehydroepiandrosterone, and dehydroepiandrosterone sulfate in serum samples from 455 reproductive-aged women (18–45 yrs) in Guangxi, China. Age-dependent concentration trends were analyzed, and reference intervals stratified by age (2.5th to 97.5th percentiles) were established. Correlations with body-composition metrics, ethnicity, and the menstrual cycle were investigated. Results: The HPLC-MS/MS method demonstrated high precision (intra- and inter-assay CVs < 15%), accuracy, and sensitivity. All eight hormones exhibited significant age-related declines (P < 0.001 for seven hormones; P = 0.001 for 11-OH A4). Notably, 11-OH A4 levels were significantly lower in the 35–45-yr (3.05 nmol/L) and 25–34-yr (3.09 nmol/L) age groups than in the 18–24-yr (3.57 nmol/L) age group, whereas no significant difference was observed between the 35–45-yr and 25–34-yr age groups. Weak negative correlations were observed between the body mass index and corticosterone and cortisone levels, whereas ethnicity and the menstrual cycle showed no significant associations with hormone levels. Conclusions We developed an HPLC-MS/MS-based method for simultaneously quantifying eight adrenocortical hormones, including 11-OH A4, and defined age-specific reference intervals for reproductive-aged Chinese women. These findings advance the clinical utility of adrenocortical hormones in diagnosing and managing reproductive disorders.

Currently, researchers have identified several mutated genes associated with hereditary eye diseases; however, effective therapeutic options remain scarce. The emergence of clustered regularly interspaced short palindromic repeats(CRISPR)and its associated proteins(CRISPR-associated proteins, Cas)offers a promising approach for treating these diseases. CRISPR/Cas9 enables precise targeting and modification of specific genetic sequences, allowing for the correction of mutated genes, as well as knockout or replacement of pathogenic genes to achieve therapeutic effects. In ophthalmology, CRISPR/Cas9 has been applied to various hereditary eye disorders, including corneal dystrophy, congenital cataracts, glaucoma, and retinitis pigmentosa. Additionally, significant progress has been made to utilize CRISPR/Cas9 to develop disease models. Therefore, it has great potential for clinical applications. However, challenges such as delivery efficiency and off-target effects remain. This review summarizes the mechanism of CRISPR/Cas9, its applications in genetic eye diseases and disease models, as well as the existing challenges, aiming to provide new insights for treatment.

Objective To evaluate the forensic application value of used dental floss as a source of bio-logical evidence for individual identification by analyzing the effects of dental floss sample collection methods,DNA extraction methods,preservation conditions,and sampling sites on the success rate of STR typing.Methods Dental floss samples were collected using three techniques:direct cutting,cotton swab wiping,and flocked swab wiping,respectively.DNA was extracted respectively by the Chelex,spin column-based and magnetic bead-based methods.DNA quantification and STR typing were per-formed using the Qubit kit and FGI HumDNA Typing kit(Platinum),respectively.Storage environ-ments(temperature and humidity,ultraviolet radiation)and sampling locations(the floss part,the handle part)on DNA quantity and STR typing were evaluated.Results Through conducting a statisti-cal analysis of three key indicators of average DNA mass concentration,STR locus detection rate,and typing accuracy rate,the direct cutting method demonstrated the highest efficacy,followed by cotton swab wiping mothed,and the flocked swab wiping method had the lowest efficacy.Direct cutting yielded an average DNA mass concentration greater than(4.94±1.87)ng/μL,with STR locus detection and accuracy rates of 100%.Bead-based DNA extraction method produced superior DNA concentration and quality compared to spin column-based and Chelex methods,regardless of whether the sampling technique used.Preservation conditions had a significant impact on the DNA analysis of samples.Par-ticularly,the STR typing accuracy of samples preserved at 55℃/50%RH for 35 days dropped to(81.82±12.31)%,and that of samples exposed to ultraviolet radiation for 12 h dropped to(55.46±34.31)%.DNA concentration from the handle part of dental floss was extremely low,with an STR typing accuracy of only(30.91±27.35)%.Conclusion Using cotton swabs to wipe or directly cutting the thread of dental floss samples,and combining this approach with the magnetic bead method for DNA extraction,can best guarantee the concentration and quality of DNA.In addition,samples should be stored in low-temperature,low-humidity environment,protected from light and ultraviolet radiation.

Objective:To explore the characteristics of social skills and problem behaviors of children and adolescents with autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), as well as the association with core symptoms.Methods:A total of 409 patients aged 5-18 years old with ASD or ADHD in the outpatient department of Nanjing Brain Hospital from 2023 to 2024, and 344 children and adolescents with typical development(TD) were recruited.All participants were matched in a ratio of 1∶1∶1 (ASD∶ADHD∶TD) according to gender and age, and 97 participants were included in each group for analysis.The Chinese version of the social skills improvement system rating scales(SSIS-RS-C) was used to evaluate social skills and problem behaviors, and autistic child behavior checklist(ABC), childhood autism rating scale(CARS), the Chinese version of the social communication questionnaire(SCQ) and the Chinese version of Swanson, Nolan, and Pelham, version Ⅳ scale-parent form(SNAP-Ⅳ) were used to evaluate the core symptoms of ASD and ADHD, respectively. SPSS 26.0 software was used to perform variance, Chi-square test, Spearman correlation analysis and multivariate Logistic regression analysis.Results:The social skills score of ASD group was lower than ADHD group ((61.53±24.26) vs (80.89±15.19), P<0.05), while the problem behavior score of ASD group was higher than ADHD group ((38.82±11.92) vs (34.00±12.45), P<0.05). In ASD group, the scores of ABC, CARS and SCQ were negatively correlated with the score of social skills ( r=-0.26--0.55, P<0.05). In ADHD group, the total score and each subscale of SNAP-Ⅳ were positively correlated with the score of problem behavior ( r=0.25-0.65, P<0.05). Multivariate Logistic regression analysis showed that empathy was a negative influencing factor of ASD ( B=-0.246, OR=0.782, P<0.05), and hyperactivity/inattention was a positive influencing factor of ASD ( B=0.589, OR=1.802, P<0.01), while only hyperactivity/inattention was a positive influencing factor of ADHD( B=0.779, OR=2.180, P<0.01). Conclusion:Children and adolescents with ASD and ADHD both have defects in social skills and problem behaviors, and these defects are associated with the core characteristics of their respective diseases.

Purpose To investigate the correlation between intervertebral disc degeneration and paraspinal muscle fatty infiltration in chronic low back pain(CLBP)using quantitative MRI techniques.Materials and Methods A total of 63 CLBP patients and 42 volunteers in Zigong Fourth People's Hospital from September 2023 to March 2024 were retrospectively collected.Magnetic resonance scans were performed using multi-echo water-fat separation technique and T2 mapping technique.Intervertebral disc degeneration was assessed using the Pfirrmann grading,while paraspinal muscle fatty infiltration was evaluated using the Goutallier grading.Proton density fat fraction(PDFF)of the multifidus and erector spinae muscles,and T2 values of the nucleus pulposus were quantitatively measured.The relationships between these parameters and their associations with age and gender were analyzed.Results Compared with healthy volunteers,CLBP patients showed significantly reduced T2 values of the nucleus pulposusand significantly increased PDFF of the paraspinal muscles(Z=-5.54-4.65,all P<0.001).The Pfirrmann grading of intervertebral discs and the Goutallier grading of paraspinal muscles were significantly correlated with the T2 value of the nucleus pulposus and the PDFF of the paraspinal muscles(|r|>0.4,P<0.05).Age and Pfirrmann grading were independent predictors of paraspinal muscle fat infiltration(β=0.228-5.682,all P<0.001),and gender was also an independent predictor at the L4/5 intervertebral disc level(β=-2.834,-3.352,both P<0.05).Conclusion Intervertebral disc degeneration and paraspinal muscle fat infiltration are closely related in CLBP,age and gender also have an impact.

Osteoarthritis (OA), the most prevalent joint disease of late life, is closely linked to cellular senescence. Previously, we found that the senescence of fibroblast-like synoviocytes (FLS) played an essential role in the degradation of cartilage. In this work, single-cell sequencing data further demonstrated that cartilage acidic protein 1 (CRTAC1) is a critical secreted factor of senescent FLS, which suppresses mitophagy and induces mitochondrial dysfunction by regulating SIRT3 expression. In vivo, deletion of SIRT3 in chondrocytes accelerated cartilage degradation and aggravated the progression of OA. Oppositely, intra-articular injection of adeno-associated virus expressing SIRT3 effectively alleviated OA progression in mice. Mechanistically, we demonstrated that elevated CRTAC1 could bind with NRF2 in chondrocytes, which subsequently suppresses the transcription of SIRT3 in vitro. In addition, SIRT3 reduction could promote the acetylation of FOXO3a and result in mitochondrial dysfunction, which finally contributes to the degradation of chondrocytes. To conclude, this work revealed the critical role and underlying mechanism of senescent FLSs-derived CRTAC1 in OA progression, which provided a potential strategy for the OA therapy.

Immune suppression in the tumor microenvironment (TME) is closely related to abnormal glycolysis. Tumor cells gain metabolic advantages and suppress immune responses through the "Warburg effect". Traditional Chinese medicine (TCM) has been shown to regulate key glycolysis enzymes (such as HK2 and PKM2), metabolic signaling pathways (such as PI3K/AKT/mTOR, HIF-1α) and non-coding RNAs at multiple targets, thus synergistically inhibiting lactate accumulation, improving vascular abnormalities, and relieving metabolic inhibition of immune cells. Studies have shown that TCM monomers and formulas can promote immune cell infiltration and functions, improve metabolic microenvironment, and with the assistance by the nano-delivery system, enhance the precision of treatment. However, the dynamic mechanism of the interaction between TCM-regulated glycolysis and TME has not been fully elucidated, for which single-cell sequencing and other technologies provide important technical support to facilitate in-depth analysis and clinical translational research. Future studies should be focused on the synergistic strategy of "metabolic reprogramming-immune activation" to provide new insights into the mechanisms of tumor immunotherapy.
Humans ; Tumor Microenvironment/immunology* ; Glycolysis/drug effects* ; Neoplasms/drug therapy* ; Medicine, Chinese Traditional ; Signal Transduction ; Drugs, Chinese Herbal/pharmacology*