ObjectiveJunctophilin-2 (JPH2) is an essential structural protein that maintains junctional membrane complexes (JMCs) in cardiomyocytes by tethering the plasma membrane to the sarcoplasmic reticulum, thereby facilitating excitation-contraction (E-C) coupling. Mutations in JPH2 have been associated with hypertrophic cardiomyopathy (HCM), but the molecular mechanisms governing its membrane-binding properties and the functional relevance of its membrane occupation and recognition nexus (MORN) repeat motifs remain incompletely understood. This study aimed to elucidate the structural basis of JPH2 membrane association and its implications for HCM pathogenesis. MethodsA recombinant N-terminal fragment of mouse JPH2 (residues1-440), encompassing the MORN repeats and an adjacent helical region, was purified under near-physiological buffer conditions.X-ray crystallography was employed to determine the structure of the JPH2 MORN-Helix domain. Sequence conservation analysis across species and junctophilin isoforms was performed to assess the evolutionary conservation of key structural features. Functional membrane-binding assays were conducted using liposome co-sedimentation and cell-based localization studies in COS7 and HeLa cells. In addition, site-directed mutagenesis targeting positively charged residues and known HCM-associated mutations, including R347C, was used to evaluate their effects on membrane interaction and subcellular localization. ResultsThe crystal structure of the mouse JPH2 MORN-Helix domain was resolved at 2.6 Å, revealing a compact, elongated architecture consisting of multiple tandem MORN motifs arranged in a curved configuration, forming a continuous hydrophobic core stabilized by alternating aromatic residues. A C-terminal α-helix further reinforced structural integrity. Conservation analysis identified the inner groove of the MORN array as a highly conserved surface, suggesting its role as a protein-binding interface. A flexible linker segment enriched in positively charged residues, located adjacent to the MORN motifs, was found to mediate direct electrostatic interactions with negatively charged phospholipid membranes. Functional assays demonstrated that mutation of these basic residues impaired membrane association, while the HCM-linked R347C mutation completely abolished membrane localization in cellular assays, despite preserving the overall MORN-Helix fold in structural modeling. ConclusionThis study provides structural insight into the membrane-binding mechanism of the cardiomyocyte-specific protein JPH2, highlighting the dual roles of its MORN-Helix domain in membrane anchoring and protein interactions. The findings clarify the structural basis for membrane targeting via a positively charged linker and demonstrate that disruption of this interaction—such as that caused by the R347C mutation—likely contributes to HCM pathogenesis. These results not only enhance current understanding of JPH2 function in cardiac E-C coupling but also offer a structural framework for future investigations into the assembly and regulation of JMCs in both physiological and disease contexts.

OBJECTIVE To provide a reference for optimizing and promoting the quality standards of Shechuan naolitong granules. METHODS Fifteen batches of Shechuan naolitong granules were used as samples to establish HPLC fingerprints using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine （2012 edition）. Similarity evaluation and common peak identification were performed， and orthogonal partial least squares discriminant analysis （OPLS-DA） was used to assess quality differences among different batches and to screen quality differential components. Using salvianolic acid B（SAB） as the internal reference， quantitative analysis of multi-components by single-marker （QAMS） was developed to simultaneously determine geniposidic acid （GA）， chlorogenic acid （CA）， vaccarin （VA）， ferulic acid （FA） and senkyunolide I （SI）. The results were compared with those obtained by the external standard method. RESULTS A total of 13 common peaks were identified in the HPLC fingerprints of 15 batches of samples， and the similarities of the spectra were all above 0.96. Seven chromatographic peaks were identified as GA （peak 3）， CA （peak 6）， VA （peak 8）， FA （peak 9）， SI （peak 11）， SAB（peak 12） and TA（peak 13）. OPLS-DA indicated that the differential quality markers among 15 batches were peaks 5， 11 （SI）， and 12 （SAB）.Using SAB as the internal reference， the relative correction factors for GA， CA， VA， FA and SI were calculated as 1.058 4， 0.594 3， 0.643 3， 0.342 7 and 0.262 8， respectively. The mean content of GA， CA， VA， FA， SI and SAB across the 15 batches of samples were 0.155 0， 0.085 4， 0.140 3， 0.071 8， 0.072 7， 1.276 3 mg/g， respectively， showing no significant difference compared with the ESM （P＞0.05）. CONCLUSIONS The established HPLC fingerprint and QAMS are simple， efficient and economical， providing a reference for the quality control and further development of Shechuan naolitong granules.

Purpose: The aim of this study was to investigate the feasibility of an intelligent handheld ultrasound (US) device for assisting non-expert general practitioners (GPs) in detecting carotid plaques (CPs) in community populations. Methods: This prospective parallel controlled trial recruited 111 consecutive community residents. All of them underwent examinations by non-expert GPs and specialist doctors using handheld US devices (setting A, setting B, and setting C). The results of setting C with specialist doctors were considered the gold standard. Carotid intima-media thickness (CIMT) and the features of CPs were measured and recorded. The diagnostic performance of GPs in distinguishing CPs was evaluated using a receiver operating characteristic curve. Inter-observer agreement was compared using the intragroup correlation coefficient (ICC). Questionnaires were completed to evaluate clinical benefits. Results: Among the 111 community residents, 80, 96, and 112 CPs were detected in settings A, B, and C, respectively. Setting B exhibited better diagnostic performance than setting A for detecting CPs (area under the curve, 0.856 vs. 0.749; P<0.01). Setting B had better consistency with setting C than setting A in CIMT measurement and the assessment of CPs (ICC, 0.731 to 0.923). Moreover, measurements in setting B required less time than the other two settings (44.59 seconds vs. 108.87 seconds vs. 126.13 seconds, both P<0.01). Conclusion Using an intelligent handheld US device, GPs can perform CP screening and achieve a diagnostic capability comparable to that of specialist doctors.

Purpose: The aim of this study was to investigate the feasibility of an intelligent handheld ultrasound (US) device for assisting non-expert general practitioners (GPs) in detecting carotid plaques (CPs) in community populations. Methods: This prospective parallel controlled trial recruited 111 consecutive community residents. All of them underwent examinations by non-expert GPs and specialist doctors using handheld US devices (setting A, setting B, and setting C). The results of setting C with specialist doctors were considered the gold standard. Carotid intima-media thickness (CIMT) and the features of CPs were measured and recorded. The diagnostic performance of GPs in distinguishing CPs was evaluated using a receiver operating characteristic curve. Inter-observer agreement was compared using the intragroup correlation coefficient (ICC). Questionnaires were completed to evaluate clinical benefits. Results: Among the 111 community residents, 80, 96, and 112 CPs were detected in settings A, B, and C, respectively. Setting B exhibited better diagnostic performance than setting A for detecting CPs (area under the curve, 0.856 vs. 0.749; P<0.01). Setting B had better consistency with setting C than setting A in CIMT measurement and the assessment of CPs (ICC, 0.731 to 0.923). Moreover, measurements in setting B required less time than the other two settings (44.59 seconds vs. 108.87 seconds vs. 126.13 seconds, both P<0.01). Conclusion Using an intelligent handheld US device, GPs can perform CP screening and achieve a diagnostic capability comparable to that of specialist doctors.

Purpose: The aim of this study was to investigate the feasibility of an intelligent handheld ultrasound (US) device for assisting non-expert general practitioners (GPs) in detecting carotid plaques (CPs) in community populations. Methods: This prospective parallel controlled trial recruited 111 consecutive community residents. All of them underwent examinations by non-expert GPs and specialist doctors using handheld US devices (setting A, setting B, and setting C). The results of setting C with specialist doctors were considered the gold standard. Carotid intima-media thickness (CIMT) and the features of CPs were measured and recorded. The diagnostic performance of GPs in distinguishing CPs was evaluated using a receiver operating characteristic curve. Inter-observer agreement was compared using the intragroup correlation coefficient (ICC). Questionnaires were completed to evaluate clinical benefits. Results: Among the 111 community residents, 80, 96, and 112 CPs were detected in settings A, B, and C, respectively. Setting B exhibited better diagnostic performance than setting A for detecting CPs (area under the curve, 0.856 vs. 0.749; P<0.01). Setting B had better consistency with setting C than setting A in CIMT measurement and the assessment of CPs (ICC, 0.731 to 0.923). Moreover, measurements in setting B required less time than the other two settings (44.59 seconds vs. 108.87 seconds vs. 126.13 seconds, both P<0.01). Conclusion Using an intelligent handheld US device, GPs can perform CP screening and achieve a diagnostic capability comparable to that of specialist doctors.

Purpose: The aim of this study was to investigate the feasibility of an intelligent handheld ultrasound (US) device for assisting non-expert general practitioners (GPs) in detecting carotid plaques (CPs) in community populations. Methods: This prospective parallel controlled trial recruited 111 consecutive community residents. All of them underwent examinations by non-expert GPs and specialist doctors using handheld US devices (setting A, setting B, and setting C). The results of setting C with specialist doctors were considered the gold standard. Carotid intima-media thickness (CIMT) and the features of CPs were measured and recorded. The diagnostic performance of GPs in distinguishing CPs was evaluated using a receiver operating characteristic curve. Inter-observer agreement was compared using the intragroup correlation coefficient (ICC). Questionnaires were completed to evaluate clinical benefits. Results: Among the 111 community residents, 80, 96, and 112 CPs were detected in settings A, B, and C, respectively. Setting B exhibited better diagnostic performance than setting A for detecting CPs (area under the curve, 0.856 vs. 0.749; P<0.01). Setting B had better consistency with setting C than setting A in CIMT measurement and the assessment of CPs (ICC, 0.731 to 0.923). Moreover, measurements in setting B required less time than the other two settings (44.59 seconds vs. 108.87 seconds vs. 126.13 seconds, both P<0.01). Conclusion Using an intelligent handheld US device, GPs can perform CP screening and achieve a diagnostic capability comparable to that of specialist doctors.

Based on copper death, this study investigates the effect and mechanism of Shenmai Injection on isoproterenol(ISO)-induced myocardial fibrosis(MF) in rats. SPF-grade male SD rats were randomly divided into a normal group, model group, captopril(5 mg·kg~(-1)) positive control group, and Shenmai Injection low(6 mL·kg~(-1)), medium(9 mL·kg~(-1)), and high(12 mL·kg~(-1)) dose groups. Except for the normal group, the rats in the other groups were subcutaneously injected with ISO(5 mg·kg~(-1)) once a day for 10 consecutive days to establish an MF model. Starting from the second day after successful modeling, intraperitoneal injections of the respective treatments were administered for 28 consecutive days. Hematoxylin-eosin(HE) and Masson staining were used to observe pathological changes and fibrosis levels in the myocardial tissue. Colorimetry was employed to detect serum Cu~(2+) concentration in rats. The levels of inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β), interleukin-18(IL-18), tumor necrosis factor-α(TNF-α), as well as mitochondrial energy metabolites adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in serum were measured using enzyme-linked immunosorbent assay(ELISA). Western blot was performed to detect the expression of collagen Ⅰ(Col-Ⅰ), collagen Ⅲ(Col-Ⅲ), and copper death-related proteins dihydrolipoamide acetyltransferase(DLAT), ferredoxin 1(FDX1), lipoic acid synthetase(LIAS), and heat shock protein 70(HSP70) in myocardial tissue. Immunofluorescence was used to detect the expression of DLAT, FDX1, and HSP70, while immunohistochemistry was conducted to examine the expressions of DLAT, FDX1, LIAS, and HSP70. The results showed that, compared to the model group, the myocardial structure disorder and collagen fiber deposition in the drug treatment groups were significantly improved, the cardiac index level was reduced, serum Cu~(2+), IL-6, IL-1β, IL-18, TNF-α, ADP, and AMP levels were significantly decreased, ATP levels were significantly increased, and the expressions of Col-Ⅰ, Col-Ⅲ, and HSP70 proteins in myocardial tissue were significantly reduced, while the expressions of DLAT, FDX1, and LIAS proteins were significantly elevated. In conclusion, Shenmai Injection effectively alleviates myocardial structure disorder and interstitial collagen fiber deposition in ISO-induced MF rats, promotes copper excretion, and reduces copper death in the ISO-induced rat MF model.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Myocardium/metabolism* ; Drug Combinations ; Fibrosis/metabolism* ; Copper/blood* ; Cardiomyopathies/genetics* ; Humans

This paper aimed to explore the intestinal toxicity of Euphoriae Ebracteolata Radix(EER) before and after being processed with Mongolian medicine Hezi Decoction(HZD) and the toxicity-reducing mechanism of this processing method. The intestinal toxicity in rats treated with unprocessed EER and HZD-processed EER extracts via 95% ethanol was compared. The comparison was based on several indicators, including fecal volume, serum diamine oxidase(DAO) and D-lactate(D-LA) levels, the water content of various intestinal segments and their contents, and inflammatory factor levels in intestinal segments. Tandem mass tag(TMT) quantitative proteomics technology was employed to analyze the key proteins associated with changes in intestinal toxicity between unprocessed EER and HZD-processed EER. The results indicated that compared with the blank group, unprocessed EER significantly increased the fecal volume, serum DAO and D-LA levels, water content of the ileal segment and its contents, as well as the release levels of inflammatory factors, including tumor necrosis factor(TNF-α) and interleukin-1 beta(IL-1β) in the ileal segment of rats(P<0.05), indicating that EER can cause diarrhea, increase intestinal permeability, and induce intestinal inflammation. Compared with those in the unprocessed EER group, all indicators in the HZD-processed EER group were significantly reduced(P<0.05). The TMT quantitative proteomics analysis revealed that a total of 6 487 proteins were identified in the rat ileum tissue. Compared to the blank group, 182 proteins exhibited significant changes in the unprocessed EER group, while 907 proteins in the HZD-processed EER group showed significant changes. The intersection of the differential proteins between the two groups identified 38 common proteins. Among them, the protein levels of intestinal barrier tight junction protein claudin3, squalene monooxidase(Sqle), clusterin, Na~+/H~+ exchange regulatory cofactor NHE-RF3(Pdzk1), and Y+L amino acid transporter 1(Slc7a7) exhibited significant changes before and after processing, and these changes were closely related to intestinal barrier function. Compared with the blank group, the expression of claudin3, Pdzk1, and Slc7a7 in the raw product group was significantly down-regulated(P<0.05),while the expression of Sqle and clusterin was significantly up-regulated(P<0.05).Compared with the raw product group, the expression of claudin3, Pdzk1, and Slc7a7 in the processed product group of HZD was significantly up-regulated(P<0.05), while the expression of Sqle and clusterin was significantly down-regulated(P<0.05). Western blot was used to detect the expression level of claudin 3 in the ileum of rats in each group. The results show that compared to that in the blank group, the expression level of claudin 3 in the unprocessed EER group was significantly reduced(P<0.01); compared to that in the unprocessed EER group, the expression level of claudin 3 in the HZD-processed EER group was significantly increased(P<0.01). This finding aligned with the proteomic outcomes, indicating that claudin 3 protein levels could serve as a crucial indicator for intestinal damage caused by EER. In summary, HZD-processed EER can reduce EER's intestinal toxicity, and the primary mechanism for its alleviation of intestinal barrier damage is the regulation of the intestinal barrier tight junction protein claudin 3 and other intestinal-related proteins.
Animals ; Drugs, Chinese Herbal/adverse effects* ; Proteomics ; Rats ; Male ; Rats, Sprague-Dawley ; Intestines/drug effects* ; Intestinal Mucosa/drug effects* ; Tumor Necrosis Factor-alpha/metabolism*

This study aims to explore the scientific connotation of sinking Rehmanniae Radix has the best quality and compare the quality between floating and sinking fresh Rehmanniae Radix samples. Ultra-performance liquid chromatography tandem quadrupole electrostatic field Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) was employed to detect the chemical components in floating and sinking fresh Rehmanniae Radix samples. The fingerprint of fresh Rehmanniae Radix was established by high performance liquid chromatography(HPLC), and four index components were determined simultaneously. The cluster analysis, principal component analysis(PCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were conducted to compare the quality of floating and sinking fresh Rehmanniae Radix samples. An evaporative light-scattering detector was used to compare the content of five sugars. The extract yield and drying rate were determined, and the quality connotation of sinking Rehmanniae Radix has the best quality was explained by multiple indicators. A total of 41 components were preliminarily identified from fresh Rehmanniae Radix by UHPLC-Q-Orbitrap HRMS, including 7 iridoid glycosides, 9 phenylethanol glycosides, 6 amino acids, 4 sugars, 3 phenolic acids, 5 nucleosides, 3 organic acids, 1 ionone, 1 furan, 1 coumarin, and 1 phenylpropanoid. The results showed that the main chemical components were consistent between floating and sinking fresh Rehmanniae Radix. Nine common peaks were identified in the fingerprints of 15 batches of floating and sinking fresh Rehmanniae Radix samples, and the similarity of fingerprints was greater than 0.9. The cluster analysis, PCA, and OPLS-DA classified floating and sinking fresh Rehmanniae Radix sasmples into two categories, indicating differences in the quality between them. The total content of catalpol, rehmannioside D, ajugol, and verbascoside in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was catalpol. The total content of fructose, glucose, sucrose, raffinose, and stachyose in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was stachyose. The extract yield and drying rate of the sinking samples were higher than those of floating samples. This study preliminarily showed that floating and sinking fresh Rehmanniae Radix samples had the same components but great differences in the content of medicinal substance basis. The total content of four glycosides and five sugars, extract yield, and drying rate of sinking fresh Rehmanniae Radix samples is higher than that of floating samples of the same batch and specification. These findings, to a certain extent, explains the scientificity of sinking Rehmanniae Radix has the best quality recorded in ancient books and provide a reference for the quality control and clinical application of fresh Rehmanniae Radix.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Rehmannia/chemistry* ; Chemometrics ; Mass Spectrometry/methods* ; Quality Control ; Principal Component Analysis ; Plant Extracts

The quality of traditional Chinese medicine(TCM) is a critical foundation for ensuring the stability of its efficacy, as well as the safety and effectiveness of its clinical use. The identification of critical quality attributes(CQAs) is one of the core components of TCM preparation quality control. This study focuses on Jianwei Xiaoshi Tablets and explores their CQAs related to property and flavor from the perspective of taste receptor proteins. Three taste receptor proteins, T1R2, T1R3, and TRPV1, were selected, and a biosensor based on high-electron-mobility transistor(HEMT) was constructed to detect the interactions between Jianwei Xiaoshi Tablets and taste receptor proteins. Simultaneously, liquid chromatography-mass spectrometry(LC-MS) technology was used to analyze the chemical composition of Jianwei Xiaoshi Tablets. In examining the interaction strength, the results indicated that the interaction between Jianwei Xiaoshi Tablets and TRPV1 protein was the strongest, followed by T1R3, with the interaction with T1R2 being relatively weaker. By combining biosensing technology with LC-MS, 16 chemical components were identified from Jianwei Xiaoshi Tablets, among which six were selected as CQAs for sweetness and seven for pungency. Further validation experiments demonstrated that CQAs such as hesperidin and hesperetin had strong interactions with their corresponding taste receptor proteins. Through the combined use of multiple technological approaches, this study successfully determined the property and flavor-related CQAs of Jianwei Xiaoshi Tablets. It provides novel ideas and approach for the identification of CQAs in TCM preparations and offers comprehensive theoretical support for TCM quality control, contributing to the improvement and development of TCM preparation quality control systems.
Drugs, Chinese Herbal/chemistry* ; Biosensing Techniques/methods* ; TRPV Cation Channels/chemistry* ; Tablets/chemistry* ; Receptors, G-Protein-Coupled/genetics* ; Quality Control ; Taste ; Humans ; Mass Spectrometry