ObjectiveTo systematically evaluate the content differences of 4 components in Leonuri Herba granules， reveal the quality fluctuation patterns of products from the same and different manufacturers， providing scientific basis for the optimization of production process and quality control. MethodsHigh performance liquid chromatography-diode array detector-charged aerosol detector（HPLC-DAD-CAD） was employed to determine the contents of 4 components（syringic acid， leonurine hydrochloride， ferulic acid， and stachydrine hydrochloride） in samples from 19 manufacturers（53 batches， 159 boxes）. Additionally， fingerprint profiles were constructed， and the fingerprint dissimilarity（P_S） and relative standard deviation（RSD） of different samples from the same manufacturer were calculated. A principal component analysis（PCA） model was established with P_S and the RSD values of the 4 components as variables to classify the manufacturers. Finally， samples from 5 manufacturers（M1-M5） covering three consistency groups were selected to calculate three quality consistency parameters， namely intra-batch consistency（P_A）， inter-batch consistency（P_B）， and P_S. Then， PCA was performed with P_A， P_B， and P_S of these 5 manufacturers as variables. ResultsThe average total content of the 4 index components per bag across the 19 manufacturers ranged from 41.10 mg to 97.54 mg. Among them， the content of stachydrine hydrochloride（a pharmacopoeial quality control component） was 32.46-72.70 mg per bag， all meeting the requirements of the 2025 edition of the Pharmacopoeia of the People's Republic of China， with RSD of 1.7%-17.1%. The content ranges of the other 3 components were as follows：syringic acid of 1.43-41.92 mg per bag， leonurine hydrochloride of 0.67-11.85 mg per bag， and ferulic acid of 0.11-3.81 mg per bag. Notably， leonurine hydrochloride exhibited the most significant content fluctuation among samples from the same manufacturer（RSD of 4.8%-59.2%）. PCA results showed that the 19 manufacturers could be classified into 3 categories. Samples from 8 manufacturers（M2， M6， M7， M8， M10， M15， M17， M18） demonstrated relatively high consistency， five manufacturers（M3， M9， M12， M13， M14） showed moderate consistency， six manufacturers（M1， M4， M5， M11， M16， M19） exhibited low consistency. The two methods yielded consistent classification results for the 5 representative manufacturers， verifying the reliability of the proposed method. Among these， manufacturer M2 showed the best quality consistency and the highest total content of indicator components among M1-M5. ConclusionThe HPLC-DAD-CAD multi-detector hyphenation technology established in this study enables the accurate detection of 4 components in Leonuri Herba granules. Significant differences in the total content of these four components are observed among products from 19 manufacturers. The application of 2 consistency evaluation methods combined with PCA can effectively classify their consistency into 3 categories， and the classification results of the 2 methods are highly consistent. This study provides scientific basis for the process optimization and quality standard improvement of Leonuri Herba granules.

ObjectiveTo systematically evaluate the content differences of 4 components in Leonuri Herba granules， reveal the quality fluctuation patterns of products from the same and different manufacturers， providing scientific basis for the optimization of production process and quality control. MethodsHigh performance liquid chromatography-diode array detector-charged aerosol detector（HPLC-DAD-CAD） was employed to determine the contents of 4 components（syringic acid， leonurine hydrochloride， ferulic acid， and stachydrine hydrochloride） in samples from 19 manufacturers（53 batches， 159 boxes）. Additionally， fingerprint profiles were constructed， and the fingerprint dissimilarity（P_S） and relative standard deviation（RSD） of different samples from the same manufacturer were calculated. A principal component analysis（PCA） model was established with P_S and the RSD values of the 4 components as variables to classify the manufacturers. Finally， samples from 5 manufacturers（M1-M5） covering three consistency groups were selected to calculate three quality consistency parameters， namely intra-batch consistency（P_A）， inter-batch consistency（P_B）， and P_S. Then， PCA was performed with P_A， P_B， and P_S of these 5 manufacturers as variables. ResultsThe average total content of the 4 index components per bag across the 19 manufacturers ranged from 41.10 mg to 97.54 mg. Among them， the content of stachydrine hydrochloride（a pharmacopoeial quality control component） was 32.46-72.70 mg per bag， all meeting the requirements of the 2025 edition of the Pharmacopoeia of the People's Republic of China， with RSD of 1.7%-17.1%. The content ranges of the other 3 components were as follows：syringic acid of 1.43-41.92 mg per bag， leonurine hydrochloride of 0.67-11.85 mg per bag， and ferulic acid of 0.11-3.81 mg per bag. Notably， leonurine hydrochloride exhibited the most significant content fluctuation among samples from the same manufacturer（RSD of 4.8%-59.2%）. PCA results showed that the 19 manufacturers could be classified into 3 categories. Samples from 8 manufacturers（M2， M6， M7， M8， M10， M15， M17， M18） demonstrated relatively high consistency， five manufacturers（M3， M9， M12， M13， M14） showed moderate consistency， six manufacturers（M1， M4， M5， M11， M16， M19） exhibited low consistency. The two methods yielded consistent classification results for the 5 representative manufacturers， verifying the reliability of the proposed method. Among these， manufacturer M2 showed the best quality consistency and the highest total content of indicator components among M1-M5. ConclusionThe HPLC-DAD-CAD multi-detector hyphenation technology established in this study enables the accurate detection of 4 components in Leonuri Herba granules. Significant differences in the total content of these four components are observed among products from 19 manufacturers. The application of 2 consistency evaluation methods combined with PCA can effectively classify their consistency into 3 categories， and the classification results of the 2 methods are highly consistent. This study provides scientific basis for the process optimization and quality standard improvement of Leonuri Herba granules.

AIM: To explore the effect of adiponectin(ADPN)on angiogenesis of human retinal microvascular endothelial cells(hRMECs)in high glucose(HG)environment and role of NOD-like receptor family pyrin domain containing 3(NLRP3)inflammasome.METHODS: The hRMECs were divided into six groups, including control group(without treatment), HG group: incubated with D-glucose, ADPN group: pretreatment with ADPN and then incubated with D-glucose, CY-09 group: pretreatment with CY-09(an NLRP3 inhibitor)and then incubated with D-glucose, Nigericin group: pretreatment with nigericin(an NLRP3 activator)and then incubated with D-glucose, Nigericin+ADPN group: pretreatment with nigericin and ADPN and then incubated with D-glucose. NLRP3 level was detected using Western blot analysis. hRMECs migration was measured using scratch wound healing assay. The tube formation of hRMECs was detected using Matrigel.RESULTS: The NLRP3 expression in hRMECs cultured in an HG environment was significantly increased(P<0.01), while ADPN and CY-09 reduced the elevated NLRP3(both P<0.05 vs HG group). Nigericin significantly increased NLRP3 levels(P<0.01 vs control group)which was reversed by ADPN(P=0.032 vs Nigericin group). hRMECs migration ability(P<0.001), and total master segments length and number of meshes increased in HG group(P<0.001)while decreased in ADPN and CY-09 groups(all P<0.01 vs HG group). The hRMECs migration ability and tube formation(total master segments length and number of meshes)in HG environment were significantly increased by nigericin(P=0.003), while ADPN inversed the change. CONCLUSION: ADPN alleviates the migration and angiogenesis of hRMECs under HG conditions.

ObjectiveTo investigate the disease burden of non-alcoholic fatty liver disease （NAFLD） among young people in East Asian countries （including China， North Korea， Japan， Mongolia， and South Korea） in 1990—2023， and to provide a basis for formulating and adjusting the prevention and treatment strategies for NAFLD. MethodsRelated disease burden data of NAFLD among the young people in these five East Asian countries were collected from the Global Burden of Disease 2023 （GBD 2023） database， including incidence rate， prevalence rate， and disability-adjusted life years （DALY）， and the young people were divided into groups based on country， sex， and age. Estimated annual percentage change （EAPC） was used to comprehensively evaluate the changing trend of the disease burden of NAFLD among young people from 1990 to 2023， and the Autoregressive Integrated Moving Average model was used to predict the disease burden of NAFLD among the young people in China. ResultsIn 2023， the standardized incidence rates of NAFLD among the young people in China， North Korea， Japan， Mongolia， and South Korea were 926.57/100 000， 805.83/100 000， 412.66/100 000， 747.66/100 000， and 534.08/100 000， respectively； the standardized prevalence rates of NAFLD among the young people in China， North Korea， Japan， Mongolia， and South Korea were 16 817.94/100 000， 14 500.98/100 000， 8 534.40/100 000， 13 705.28/100 000， and 11 587.93/100 000， respectively； the standardized DALY rates of NAFLD among the young people in China， North Korea， Japan， Mongolia， and South Korea were 6.34/100 000， 10.18/100 000， 5.23/100 000， 47.93/100 000， and 6.76/100 000， respectively. From 1990 to 2023， there was a tendency of increase in the standardized incidence rate of NAFLD among the young people in China， North Korea， Japan， Mongolia， and South Korea， with an EAPC of 0.87%， 0.52%， 0.26%， 0.48%， and 0.87%， respectively； there was also a tendency of increase in the standardized prevalence rate of NAFLD among the young people in China， North Korea， Japan， Mongolia， and South Korea， with an EAPC of 0.99%， 0.49%， 0.24%， 0.59%， and 0.93%， respectively； conversely， there was a tendency of reduction in the standardized DALY rate of NAFLD among the young people in China， Japan， Mongolia， and South Korea， with an EAPC of -2.21%， -2.01%， -0.37%， and -4.62%， respectively， while the standardized DALY rate of NAFLD among the young people in North Korea remained relatively stable， with an EAPC of -0.01%. The subgroup analysis based on sex showed that there was a significant difference in the disease burden of NAFLD between the young people with different sexes in the five East Asian countries， and the subgroup analysis based on age showed that the incidence rate of NAFLD in young people decreased with the increase in age， while the prevalence rate and DALY rate of NAFLD increased with age. In addition， the standardized incidence rate and standardized prevalence rate of NAFLD among Chinese young people would further increase in the future， while there would be a reduction in standardized DALY rate in the future. ConclusionThere is a significant difference in the disease burden of NAFLD in young people across different countries， sexes， and age groups. In the future， targeted prevention and treatment measures should be developed based on the distribution characteristics of the disease burden of NAFLD， in order to effectively reduce the disease burden of NAFLD among young people.

OBJECTIVE To investigate the clinical characteristics of euglycemic diabetic ketoacidosis （euDKA） induced by empagliflozin， and provide references for safe medication. METHODS Case reports of euDKA induced by empagliflozin were retrieved and analyzed from PubMed， Web of Science， CNKI， and Wanfang Data. The Naranjo’s scale was adopted to assess the causal relationship between empagliflozin and euDKA. RESULTS A total of 29 patients were enrolled， including 16 males and 13 females，with a median age of 58 years. All patients were diagnosed with type 2 diabetes mellitus； 22 patients had at least one comorbidity. The median time to euDKA onset was 60 days after administration， and 7 cases occurred within 2 weeks of treatment. Perioperative periods，fasting，and low-carbohydrate or ketogenic diets were the main predisposing factors. Common presentations included nausea，vomiting， fatigue， tachypnea，and tachycardia. The causal relationship between empagliflozin and euDKA was assessed as “probable” in all cases. According to adverse reaction severity grading， 13 cases were grade 3 （severe） and 16 cases were grade 4 （life-threatening）. All patients discontinued empagliflozin and recovered after corresponding treatment. CONCLUSIONS euDKA is a serious adverse reaction to empagliflozin， which mostly occurs early in treatment. Perioperative periods， fasting， and ketogenic diets are prone to induce euDKA. Elderly patients and those with comorbidities are at higher risk. Risk assessment should be conducted before clinical administration； blood ketones and acid-base monitoring should be strengthened in the early medication period； withhold the drug before elective surgery， avoid prolonged fasting；upon diagnosis， discontinue empagliflozin and initiate intravenous insulin， provide fluid resuscitation promptly， and use other treatments to improve patient prognosis.

This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.
Animals ; Mice, Inbred BALB C ; RNA, Circular ; Mice ; Humans ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Antibodies, Viral/blood* ; HEK293 Cells ; Female ; Nanoparticles ; Plasmids

Objective:To explore the expression changes of nerve growth factor (NGF)/tropomyosin receptor kinase A (TrkA) signaling pathway of dorsal root ganglia (DRG) in incisional rat remifentanil-induced hyperalgesia and its effect on the expression of membrane delta opioid receptor (DOR).Methods:A total of 48 SPF male SD rats were randomly divided into 6 groups based on body weight matching, with 8 in each group, which were control group (infusion of 0.9% NaCl solution via the tail vein), incision pain group (incision pain model established using the Brennan method), remifentanil group (infusion of remifentanil via the tail vein), incision pain+ remifentanil model group (incision pain model established using the Brennan method, followed by infusion of remifentanil via the tail vein), NGF group and TrkA inhibitor group(established incision pain+ remifentanil model after intrathecal injection of NGF (0.06 μg/g) or K252a (0.3 μg/g, TrkA inhibitor)). Mechanical paw withdrawal threshold (PWT) was used to assess pain sensitivity in rats. Western blot was employed to measure the expression of NGF, TrkA, and the total DOR(tDOR) and the membrane DOR(mDOR) in DRG tissues. Immunoelectron microscopy was used to detect subcellular DOR expression in DRG. Data were processed using SPSS 24.0 software. Multiple comparisons among groups were conducted by repeated measures ANOVA or one-way ANOVA, and post-hoc comparisons were performed using the Bonferroni test.Results:(1) The results of pain behavior showed that there was a significant interaction effect between time and group in the comparison of PWT among the six groups of rats before and after intervention ( F=345.817, P<0.001). At each time point after intervention, the PWTs of the incision pain+ remifentanil group were lower than those of the incision pain group and remifentanil group, the PWTs of the NGF group were lower than those of the incision pain+ remifentanil group, and the PWTs of the TrkA inhibitor group were higher than those of the incision pain+ remifentanil group and NGF group (all P<0.05). (2)The Western blot results showed that there were statistically significant differences in the relative levels of NGF, TrkA, and mDOR in the DRG tissues of the six groups of rats ( F=156.2, 163.8, 421.2, all P<0.001). The levels of NGF, TrkA, and mDOR proteins in the incision pain+ remifentanil group (1.45±0.07, 1.46±0.04, 3.01±0.20) were higher than those in the incision pain group (1.25±0.05, 1.24±0.04, 1.84±0.05) and remifentanil group (1.24±0.04, 1.26±0.03, 1.84±0.04) (all P<0.05). The levels of NGF, TrkA, and mDOR in the NGF group (1.57±0.03, 1.58±0.07, 3.74±0.25) were higher than those in the incision pain+ remifentanil group (all P<0.05). The relative expression levels of TrkA, and mDOR in the TrkA inhibitor group (1.25±0.04, 1.68±0.07) were lower than those in the incision pain+ remifentanil group and the NGF group (all P<0.05). (3)The results of immunoelectron microscopy showed that there were statistically significant differences in the localization of DOR in the cell membrane, subcellular sites of synthesis pathways, and subcellular localization of degradation pathways among the six groups of rat DRG tissues ( F=140.3, 60.63, 60.28, all P<0.01). The DOR of the synthesis pathway of incision pain+ remifentanil group was higher than that of the incision pain group and remifentanil group, while the DOR of the synthesis pathway of NGF was higher than that of the incision pain+ remifentanil group.The DOR of the synthesis pathway of TrkA inhibitor group was lower than that of the incision pain+ remifentanil group and NGF group (both P<0.05). The DOR of the degradation pathway in the incision pain+ remifentanil group was lower than that in the incision pain group and remifentanil group, the DOR of the degradation pathway in the NGF group was lower than that in the incision pain+ remifentanil group, and the DOR of the degradation pathway in the TrkA inhibitor group was higher than that in the incision pain+ remifentanil group and NGF group (both P<0.05). Conclusion:The NGF/TrkA signaling pathway is involved in rat incisional pain-remifentanil hyperalgesia by upregulating the delta opioid receptor of the dorsal root ganglia.

Objective To characterize neurological alterations associated with structural bowel damage in patients with Crohn's disease(CD)through radiomics-assisted neurophenotyping,utilizing multiparametric brain MRI.Methods This prospective study enrolled patients with CD who underwent brain MRI,MR enterography,and ileocolonoscopy within one week.The Lémann Index was used to quantitatively assess cumulative structural bowel damage.CD patients were stratified into two groups based on a cutoff value of 4.8:those with bowel damage(LI>4.8)and those without bowel damage(LI≤4.8).A neurophenotype model was developed to characterize the neural changes associated with bowel damage in CD.Key features were selected from first-order features extracted from multiparametric brain MRI in the training cohort and validated in an independent test cohort.Results The final study population comprised 109 patients,including 51 individuals with bowel damage and 58 without bowel damage.The neurophenotype model scores were 0.785(95%CI:0.506～0.945)in the bowel damage group and 0.155(95%CI:0.093～0.394)in the non-bowel damage group,showing a statistically significant difference between the two groups(P<0.001).The developed model exhibited strong discriminative performance,with area under the receiver operating characteristic curve(AUC)values ranging from 0.824 to 0.918 across the training,vali-dation,and test cohorts(all P<0.05).Conclusion Our radiomics-assisted neurophenotype analysis reveals neural alterations in CD patients with bowel damage,which may indicate extraintestinal manifestations associated with cumulative intestinal injury.

Objective:To analyze the effect of influenza vaccination on influenza cluster epidemic in primary and secondary schools in Beijing during the 2023-2024 surveillance season and provide evidence for the improvement of influenza vaccination strategies.Methods:The incidence data of influenza cluster epidemic and influenza vaccination coverage in the schools in Beijing during 2023-2024 were collected. Descriptive epidemiological methods were used to analyze cluster epidemic characteristics, and χ2 test was used to compare incidence differences between groups, and OR value and vaccine effectiveness [VE=(1- OR)×100%] were calculated. A negative binomial regression model was used to evaluate the association between school vaccination rates and cluster epidemic risk. Joinpoint regression was used to analyze trends in relative risk ( RR) with increasing vaccination coverage and to determine the optimal vaccination threshold. Results:A total of 126 influenza cluster epidemic were reported in 115 primary and secondary schools in Beijing during 2023-2024 with the median size of 15 case, the average attack rate was 36.26% (2 033/5 607). The epidemics mainly occurred in urban area (70, 55.56%). Primary schools were the main setting (78, 61.90%), and influenza A(H3N2) was the predominant subtype (108, 85.71%). The overall influenza vaccination coverage in the primary and secondary students was 54.26%, while the average vaccination in classes affected by the epidemics was 58.57%. The overall protection rate was 47.62%, the protection rate was higher in primary schools (49.65%) than in secondary schools (46.60%). The protection rates against influenza A(H1N1)pdm09 (80.93%) and influenza B (Victoria lineage) (81.65%) were significantly higher than that against influenza A(H3N2) (44.19%). When school vaccination coverage reached ≥76.00%, the epidemic risk decreased by 52.82%.Conclusions:Even the match between influenza vaccine strains and circulating strains is suboptimal, increasing influenza vaccination coverage in schools can effectively reduce the risk for influenza cluster epidemic. In the future, measures such as policy guidance and public health education should be taken to further improve vaccination coverage, thereby establishing herd immunity and reducing the transmission risk of influenza in schools.