ObjectiveThis paper aims to find the identified and validated clinical biomarker data building upon a clinical study of early-phase phase Ⅱ and investigate the correlation analysis of Huanglian Jiedu Wan on syndrome improvement and clinical biomarkers in the treatment of "excess heat-toxicity" based on a machine learning model. Additionally， the effective prediction of clinical biomarker values for the main symptoms of the "excess heat-toxicity" syndrome was assessed. MethodsA total of 229 patients meeting the inclusion criteria for "excess heat-toxicity" syndrome were randomly divided into the Huanglian Jiedu Wan group and the placebo group. Syndrome score transition matrices were constructed for the Huanglian Jiedu Wan group and the placebo group based on three main symptoms of "excess heat-toxicity" syndrome， such as oral ulcers， sore throat， and gum swelling and pain. Data from the patients with these three syndromes were also integrated for an overall analysis. The corresponding syndrome score transition matrices were further constructed to visualize symptom change trends of the patients in the two groups via heatmaps. Based on the identified and validated clinical biomarkers related to inflammation， oxidative stress， and energy metabolism in the early phase， Spearman correlation analysis was employed to analyze and evaluate the associations between clinical biomarkers and syndrome improvement. Key clinical biomarkers reflecting the effect of Huanglian Jiedu Wan were screened through the comparison of differences between groups. An extreme gradient boosting （XGBoost） algorithm was used to develop a prediction model for main symptom classification， with classification performance evaluated through 10-fold cross-validation. Feature importance analysis was applied to identify variables with the greatest contribution to the prediction result. ResultsThe syndrome transition matrix results indicated that the Huanglian Jiedu Wan group showed a superior effect to the placebo group in improving oral ulcers， sore throat， and overall symptoms， with significant effects observed especially in sore throat and overall symptom analyses （P<0.01）. Spearman correlation analysis revealed that several clinical biomarkers positively correlated with "excess heat-toxicity" syndrome and its main symptom improvement， were also called "heat-related biomarkers"， including succinic acid， α-ketoglutaric acid， glycine， lactic acid， adenosine monophosphate （AMP）， tumor necrosis factor-α （TNF-α）， interferon-γ （IFN-γ）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， interleukin-10 （IL-10）， and so on. Conversely， clinical biomarkers negatively correlated with symptom severity， were also called "heat-clearing related biomarkers" after administration of Huanglian Jiedu Wan， including malic acid， fumaric acid， cis-aconitic acid， adrenocorticotropic hormone （ACTH）， IL-1β， IL-4， IL-8， succinic acid， and citric acid. The XGBoost classification model using all 52 biomarkers as variables achieved an average test accuracy of 0.754 and an average F1 score of 0.777. Feature importance analysis identified the scores of glutamic acid in saliva and IL-6 were the highest in all the variables， with importance scores of 0.081 and 0.080， respectively. After screening out 14 key variables and optimizing the parameters， model performance improved to an average accuracy of 0.758 and an F1 score of 0.798. Feature importance analysis further determined that the glutamic acid in saliva and IL-6 showed obvious changes after screening the variables， confirming the good syndrome prediction ability of the model constructed by these key clinical biomarkers. ConclusionThis study systematically elucidates the correlation between syndrome improvement and clinical biomarkers of Huanglian Jiedu Wan in the treatment of "excess heat-toxicity" syndrome. An XGBoost classification model based on key clinical biomarkers is successfully established， achieving effective prediction of the symptoms related to the "excess heat-toxicity" syndrome such as oral ulcers and sore throat and providing a new insight for objective identification of traditional Chinese medicine syndromes.

ObjectiveThis paper aims to find the identified and validated clinical biomarker data building upon a clinical study of early-phase phase Ⅱ and investigate the correlation analysis of Huanglian Jiedu Wan on syndrome improvement and clinical biomarkers in the treatment of "excess heat-toxicity" based on a machine learning model. Additionally， the effective prediction of clinical biomarker values for the main symptoms of the "excess heat-toxicity" syndrome was assessed. MethodsA total of 229 patients meeting the inclusion criteria for "excess heat-toxicity" syndrome were randomly divided into the Huanglian Jiedu Wan group and the placebo group. Syndrome score transition matrices were constructed for the Huanglian Jiedu Wan group and the placebo group based on three main symptoms of "excess heat-toxicity" syndrome， such as oral ulcers， sore throat， and gum swelling and pain. Data from the patients with these three syndromes were also integrated for an overall analysis. The corresponding syndrome score transition matrices were further constructed to visualize symptom change trends of the patients in the two groups via heatmaps. Based on the identified and validated clinical biomarkers related to inflammation， oxidative stress， and energy metabolism in the early phase， Spearman correlation analysis was employed to analyze and evaluate the associations between clinical biomarkers and syndrome improvement. Key clinical biomarkers reflecting the effect of Huanglian Jiedu Wan were screened through the comparison of differences between groups. An extreme gradient boosting （XGBoost） algorithm was used to develop a prediction model for main symptom classification， with classification performance evaluated through 10-fold cross-validation. Feature importance analysis was applied to identify variables with the greatest contribution to the prediction result. ResultsThe syndrome transition matrix results indicated that the Huanglian Jiedu Wan group showed a superior effect to the placebo group in improving oral ulcers， sore throat， and overall symptoms， with significant effects observed especially in sore throat and overall symptom analyses （P<0.01）. Spearman correlation analysis revealed that several clinical biomarkers positively correlated with "excess heat-toxicity" syndrome and its main symptom improvement， were also called "heat-related biomarkers"， including succinic acid， α-ketoglutaric acid， glycine， lactic acid， adenosine monophosphate （AMP）， tumor necrosis factor-α （TNF-α）， interferon-γ （IFN-γ）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， interleukin-10 （IL-10）， and so on. Conversely， clinical biomarkers negatively correlated with symptom severity， were also called "heat-clearing related biomarkers" after administration of Huanglian Jiedu Wan， including malic acid， fumaric acid， cis-aconitic acid， adrenocorticotropic hormone （ACTH）， IL-1β， IL-4， IL-8， succinic acid， and citric acid. The XGBoost classification model using all 52 biomarkers as variables achieved an average test accuracy of 0.754 and an average F1 score of 0.777. Feature importance analysis identified the scores of glutamic acid in saliva and IL-6 were the highest in all the variables， with importance scores of 0.081 and 0.080， respectively. After screening out 14 key variables and optimizing the parameters， model performance improved to an average accuracy of 0.758 and an F1 score of 0.798. Feature importance analysis further determined that the glutamic acid in saliva and IL-6 showed obvious changes after screening the variables， confirming the good syndrome prediction ability of the model constructed by these key clinical biomarkers. ConclusionThis study systematically elucidates the correlation between syndrome improvement and clinical biomarkers of Huanglian Jiedu Wan in the treatment of "excess heat-toxicity" syndrome. An XGBoost classification model based on key clinical biomarkers is successfully established， achieving effective prediction of the symptoms related to the "excess heat-toxicity" syndrome such as oral ulcers and sore throat and providing a new insight for objective identification of traditional Chinese medicine syndromes.

GPR126, also known as ADGRG6, is one of the most deeply studied aGPCRs. Initially, GPR126 was thought to be a receptor associated with muscle development and was primarily expressed in the muscular and skeletal systems. With the deepening of research, it was found that GPR126 is expressed in multiple mammalian tissues and organs, and is involved in many biological processes such as embryonic development, nervous system development, and extracellular matrix interactions. Compared with other aGPCRs proteins, GPR126 has a longer N-terminal domain, which can bind to ligands one-to-one and one-to-many. Its N-terminus contains five domains, a CUB (complement C1r/C1s, Uegf, Bmp1) domain, a PTX (Pentraxin) domain, a SEA (Sperm protein, Enterokinase, and Agrin) domain, a hormone binding (HormR) domain, and a conserved GAIN domain. The GAIN domain has a self-shearing function, which is essential for the maturation, stability, transport and function of aGPCRs. Different SEA domains constitute different GPR126 isomers, which can regulate the activation and closure of downstream signaling pathways through conformational changes. GPR126 has a typical aGPCRs seven-transmembrane helical structure, which can be coupled to Gs and Gi, causing cAMP to up- or down-regulation, mediating transmembrane signaling and participating in the regulation of cell proliferation, differentiation and migration. GPR126 is activated in a tethered-stalk peptide agonism or orthosteric agonism, which is mainly manifested by self-proteolysis or conformational changes in the GAIN domain, which mediates the rapid activation or closure of downstream pathways by tethered agonists. In addition to the tethered short stem peptide activation mode, GPR126 also has another allosteric agonism or tunable agonism mode, which is specifically expressed as the GAIN domain does not have self-shearing function in the physiological state, NTF and CTF always maintain the binding state, and the NTF binds to the ligand to cause conformational changes of the receptor, which somehow transmits signals to the GAIN domain in a spatial structure. The GAIN domain can cause the 7TM domain to produce an activated or inhibited signal for signal transduction, For example, type IV collagen interacts with the CUB and PTX domains of GPR126 to activate GPR126 downstream signal transduction. GPR126 has homology of 51.6%-86.9% among different species, with 10 conserved regions between different species, which can be traced back to the oldest metazoans as well as unicellular animals.In terms of diseases, GPR126 dysfunction involves the pathological process of bone, myelin, embryo and other related diseases, and is also closely related to the occurrence and development of malignant tumors such as breast cancer and colon cancer. However, the biological function of GPR126 in various diseases and its potential as a therapeutic target still needs further research. This paper focuses on the structure, interspecies differences and conservatism, signal transduction and biological functions of GPR126, which provides ideas and references for future research on GPR126.

Objective: To investigate the effect of integrin α5 on the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in periodontal ligament fibroblasts (PDLFs) within an inflammatory microenvironment. Methods: This study was approved by the Ethics Committee of Laboratory animals. After rat PDLFs were treated with LPS (0.5, 5, and 50 µg/mL) for 24 h, the primary medium was discarded and replaced with serum-free culture medium. After 24 h, the supernatant was collected and mixed with DMEM medium containing 10% exosome-free serum at a volume ratio of 1:1 to obtain conditioned medium (CM). The groups were labeled as the 0.5-CM, 5-CM, and 50-CM groups. In addition, PDLFs cultured in DMEM medium containing 10% exosome-free serum were considered the 0-CM group. PDLFs were cultured with the above CM. In the inhibitor group, PDLFs were cultured in 0-CM containing different concentrations of integrin α5 inhibitor ATN-161 (0, 0.025, 0.25, 2.5, 25, and 250 μg/mL). The effect of CM and integrin α5 inhibitor ATN-161 on cell viability was assessed using the CCK-8 assay. According to the CCK-8 results, in further inhibitor intervention experiments, PDLFs were cultured in 0-CM, 5-CM (without/with 25 μg/mL ATN-161), and 0-CM containing 25 μg/mL ATN-161, which were labeled as the 0-CM, 5-CM, ATN-161+5-CM, and ATN-161 groups, respectively. The expression changes of integrin α5 and NLRP3 were detected using Western blot and qRT-PCR techniques. For in vivo experiments, 24 rats were randomly divided into four groups (n=6). The control group contained healthy rats that received no treatment. The rats in the other three groups were injected with 40 µL of 0-CM containing 25 μg/mL ATN-161 or 5-CM (without or with 25 μg/mL ATN-161) on the palatal side of the left maxillary first molar every three days; these groups were classified as the ATN-161, 5-CM, and ATN-161+5-CM groups, respectively. On the 30th day, the left maxillary tissue of rats was used for Micro-CT, HE staining, and immunohistochemical detection. Results : The CCK-8 assay showed that CM, 25 μg/mL ATN-161, and ATN-161 concentrations below 25 μg/mL had no significant effect on cell viability at 12 h and 24 h (P > 0.05). 50-CM and 25 μg/mL ATN-161 significantly inhibited cell viability at 48 h (P < 0.05). For in vitro experiments, compared to the 0-CM group, both the protein and mRNA levels of integrin α5 and NLRP3 were significantly increased in rat PDLFs in the 5-CM group (P < 0.05). Intervention with 25 μg/mL ATN-161 significantly attenuated the enhancement of 5-CM on the expression of integrin α5 and NLRP3 (P < 0.05). For in vivo experiments, compared to the control group, alveolar bone resorption and periodontal inflammatory cell infiltration were significantly increased in the 5-CM and ATN-161+5-CM groups, and the expression of integrin α5 and NLRP3 was significantly increased (P < 0.01). However, compared to the 5-CM group, the ATN-161+5-CM group had less alveolar bone resorption and fewer periodontal inflammatory cells. Further, the expression of integrin α5 and NLRP3 was significantly reduced (P < 0.01). Conclusion In vitro and in vivo experiments showed that integrin α5 mediated NLRP3 expression in PDLFs under an inflammatory microenvironment. ATN-161 inhibited the expression of integrin α5, thus significantly downregulating the expression of NLRP3, which plays a role in inhibiting inflammation.

ObjectiveTo explore the dose-effect relationship and safety of high， medium， and low doses of raw Astragali Radix in the modified Huangqi Chifengtang （MHCD） for treating proteinuria in immunoglobulin A （IgA） nephropathy， and to provide scientific evidence for the clinical use of high-dose Astragali Radix in the treatment of proteinuria in IgA nephropathy. MethodsA total of 120 patients with IgA nephropathy， diagnosed with Qi deficiency and blood stasis combined with wind pathogen and heat toxicity， were randomly divided into a control group and three treatment groups. The control group received telmisartan combined with a Chinese medicine placebo， while the treatment groups were given telmisartan combined with MHCD containing different doses of raw Astragali Radix （60， 30， 15 g）. Each group contained 30 patients， and the treatment period was 12 weeks. Changes in 24-hour urinary protein （24 hUTP）， traditional Chinese medicine （TCM） syndrome scores， effective rate， and renal function were observed before and after treatment. Safety was assessed by monitoring liver function and blood routine. ResultsAfter 12 weeks of treatment， 24 hUTP significantly decreased in the high， medium， and low-dose groups， as well as the control group （P<0.05， P<0.01）. The TCM syndrome scores in the high， medium， and low-dose groups also significantly decreased （P<0.01）. Comparisons between groups showed that the 24 hUTP in the high-dose group was significantly lower than in the medium， low-dose， and control groups （P<0.05， P<0.01）， and the 24 hUTP in the medium-dose group was significantly lower than in the control group （P<0.05）. The TCM syndrome scores in the high and medium-dose groups were significantly lower than in the low-dose and control groups （P<0.05， P<0.01）. The total effective rates for proteinuria in the high， medium， low-dose， and control groups were 92.59% （25/27）， 85.19% （23/27）， 60.71% （17/28）， and 57.14% （16/28）， respectively. The effective rates in the high and medium-dose groups were significantly higher than in the low-dose and control groups （χ²=13.185， P<0.05， P<0.01）. The effective rates for TCM syndrome scores in the high， medium， low-dose， and control groups were 88.89% （24/27）， 81.48% （22/27）， 71.43% （20/28）， and 46.43% （13/28）， respectively. The efficacy of TCM syndrome scores in the high and medium-dose groups was significantly higher than in the control group （χ²=14.053， P<0.01）. Compared with pre-treatment values， there was no statistically significant difference in eGFR and serum creatinine in the high and medium-dose groups. However， eGFR significantly decreased in the low-dose and control groups after treatment （P<0.05）， and serum creatinine levels increased significantly in the control group （P<0.05）. No statistically significant differences were observed in urea nitrogen， uric acid， albumin， total cholesterol， triglycerides， liver function， and blood routine before and after treatment in any group. ConclusionThere is a dose-effect relationship in the treatment of IgA nephropathy with high， medium， and low doses of raw Astragali Radix in MHCD. The high-dose group exhibited the best therapeutic effect and good safety profile.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

BACKGROUND:Recent research indicates that disc degeneration is closely related to abnormal stress load,and mechanotransduction proteins play a key role in it. OBJECTIVE:To investigate the role and mechanism of mechanotransduction proteins in the mechanotransduction process induced by abnormal mechanical stimulation in disc degeneration,and to summarize the current treatment strategies targeting mechanotransduction to delay intervertebral disc degeneration. METHODS:Using"intervertebral disc,nucleus pulposus,annulus fibrosus,cartilaginous endplate,cell,mechanics,signal transduction,protein,biomechanics"as Chinese search terms,and"intervertebral disc,nucleus pulposus,annulus fibrosus,cartilaginous endplate,cell,mechanical stimulation,signal transduction,protein,biomechanics"as English search terms,relevant literature in the PubMed and CNKI databases was searched.A total of 88 articles were ultimately included for review. RESULTS AND CONCLUSION:Disc cells can sense external mechanical stimulation through various mechanotransduction proteins and convert it into biological responses within the cells.These transduction proteins mainly include collagen proteins in the extracellular matrix,cell membrane surface receptors(such as integrins and ion channels),and cytoskeleton structural proteins.Their regulation of mechanotransduction processes primarily involves the activation of multiple pathways,such as the PI3K/AKT signaling pathway,nuclear factor-kB signaling pathway,and Ca2+/Calpain2/Caspase3 pathway.Mechanotransduction proteins play a key role in the mechanotransduction of disc cells.Abnormal expression of these proteins or resulting changes in the extracellular matrix environment can disrupt the mechanical balance of disc cells,leading to disc degeneration.In-depth study of the expression and regulatory mechanisms of mechanotransduction proteins in disc cells,and identification of key pathological links and therapeutic targets,is of significant importance for developing treatment strategies for disc degeneration.Current strategies to delay intervertebral disc degeneration by targeting mechanotransduction mainly include regulation of transduction proteins and improvement of the extracellular matrix.However,research in this area is still in its early stages.As research continues,new breakthroughs are expected in the regulation of disc degeneration by mechanotransduction proteins.

AIM: To investigate the effect of interleukin-8(IL-8)on the regulation of monocyte chemotactic protein-1(MCP-1)secreted by lens epithelial cells(LEC)during cell migration in the development of posterior capsule opacification(PCO).METHODS: A rat lens capsule model was established and cultured in medium supplemented with 10% fetal bovine serum. Upon migration of LEC to 30%-50% of the posterior capsule, serum was removed. The capsule was subsequently divided into two groups: a control group and an IL-8(15 ng/mL)treatment group. LEC migration was captured at multiple time points. The secretion and mRNA expression of MCP-1 were quantified using ELISA and RT-qPCR, respectively. Immunofluorescence was used to assess MCP-1 expression in the different experimental groups. SRA01/04 cells were divided into three groups: control, IL-8(15 ng/mL), and IL-8(15 ng/mL)+200 μmol/L Bindarit(BND)groups, with migration measured by the Transwell assay. Additionally, SRA01/04 cells were divided into negative control(NC), NC+15 ng/mL IL-8, and 15 ng/mL IL-8+p65 siRNA groups, and MCP-1 secretion and mRNA expression were further analyzed by ELISA and RT-qPCR.RESULTS:LEC migration in the rat lens capsule cultured in vitro showed that the cells migration of the 15 ng/mL IL-8 group significantly increased at 48, 72 and 96 h(all P<0.05). ELISA results revealed that MCP-1 levels in SRA01/04 cells from the 15 ng/mL IL-8-treated group were markedly higher than those in the control group at both 12 and 24 h(all P<0.05). RT-qPCR analysis also demonstrated a significant increase in MCP-1 mRNA expression in the 15 ng/mL IL-8 group at both time points(all P<0.05). Immunofluorescence staining indicated greater MCP-1 expression in capsular epithelial cells of the 15 ng/mL IL-8 group at 24 h(P=0.007). Transwell assays further confirmed increased cell migration in the 15 ng/mL IL-8 group compared to the control group(P=0.001), while the migration reduced in the 15 ng/mL IL-8+200 μmol/L BND group compared to the 15 ng/mL IL-8 group(P=0.003). Moreover, ELISA and RT-qPCR results demonstrated a significant increase in MCP-1 secretion and mRNA expression in the NC+15 ng/mL IL-8 group at both 12 and 24 h compared to the NC group(all P<0.01). In contrast, MCP-1 secretion and mRNA expression were reduced in the 15 ng/mL IL-8+p65 siRNA group compared to the NC+15 ng/mL IL-8 group at both time points(all P<0.01).CONCLUSION: IL-8 promotes the migration of residual epithelial cells and regulates the secretion and expression of MCP-1 in LEC. The mechanism underlying IL-8's effects appears to be mediated through the activation of the NF-κB/p65 signaling pathway.

The general models for intermediates quality analysis in the production process of Yaobitong capsule were established by near infrared spectroscopy (NIRS) combined with chemometrics, realizing the rapid determination of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rd and moisture. The spray-dried fine powder and total mixed granule were selected as research objects. The contents of five saponins were determined by high performance liquid chromatography and the moisture content was determined by drying method. The measured contents were used as reference values. Meanwhile, NIR spectra were collected. After removing abnormal samples by Monte Carlo cross validation (MCCV), Monte Carlo uninformative variables elimination (MC-UVE) and competitive adaptive reweighted sampling (CARS) were used to select feature variables respectively. Based on the feature variables, quantitative models were established by partial least squares regression (PLSR), extreme learning machine (ELM) and ant lion optimization least squares support vector machine (ALO-LSSVM). The results showed that CARS-ALO-LSSVM model had the optimum effect. The correlation coefficients of the six index components were greater than 0.93, and the relative standard errors were controlled within 6%. ALO-LSSVM was more suitable for a large number of samples with rich information, and the prediction effect and stability of the model were significantly improved. The general models with good predicting effect can be used for the rapid quality determination of Yaobitong capsule intermediates.

Oral solid dosage forms require processes such as disintegration and dissolution to release the drug before it can be absorbed and utilized by the body. In this manuscript, imaging technology was used to continuously visualize and characterize the in vitro static drug release process of gliclazide modified release tablets from 15 manufacturers, combined with the traditional method of in vitro dissolution testing, to determine the release profile of gliclazide modified release tablets, to evaluate the similarity of the release profiles by using the similarity factor (f₂) method and based on the analysis of the release profiles fitted with a variety of mathematical models. The results indicate that the gliclazide modified release tablets produced by 14 companies are hydrophilic gel matrix tablets. Compared to the reference listed drug, the release profiles of formulations from 11 companies show high similarity (f₂ > 50) to the reference. Among these, formulations with visual characteristics similar to the reference exhibit similar release curves. This study provides an alternative method for the in vitro consistency evaluation of gliclazide modified release tablets, aiming to assess the in vitro release behaviour of generic formulations more accurately and comprehensively.