ObjectiveTo establish an ultra-performance liquid fingerprint and multi-components determination method for Naomaili granules. To evaluate the quality of different batches by chemometrics， and the anti-neuroinflammatory effects of water extract and main components of Naomaili granules were tested in vitro. MethodsThe similarity and common peaks of 27 batches of Naomaili granules were evaluated by using Ultra performance liquid chromatography （UPLC） fingerprint detection. Ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） technology was used to determine the content of the index components in Naomaili granules and to evaluate the quality of different batches of Naomaili granules by chemometrics. LPS-induced BV-2 cell inflammation model was used to investigate the anti-neuroinflammatory effects of the water extract and main components of Naomaili granules. ResultsThe similarity of fingerprints of 27 batches of samples was > 0.90. A total of 32 common peaks were calibrated， and 23 of them were identified and assigned. In 27 batches of Naomaili granules， the mass fractions of 14 components that were stachydrine hydrochloride， leonurine hydrochloride， calycosin-7-O-glucoside， calycosin，tanshinoneⅠ， cryptotanshinone， tanshinoneⅡ_A， ginsenoside Rb₁， notoginsenoside R₁， ginsenoside Rg₁， paeoniflorin， albiflorin， lactiflorin， and salvianolic acid B were found to be 2.902-3.498， 0.233-0.343， 0.111-0.301， 0.07-0.152， 0.136-0.228， 0.195-0.390， 0.324-0.482， 1.056-1.435， 0.271-0.397， 1.318-1.649， 3.038-4.059， 2.263-3.455， 0.152-0.232， 2.931-3.991 mg∙g^-1， respectively. Multivariate statistical analysis showed that paeoniflorin， ginsenoside Rg₁， ginsenoside Rb₁ and staphylline hydrochloride were quality difference markers to control the stability of the preparation. The results of bioactive experiment showed that the water extract of Naomaili granules and the eight main components with high content in the prescription had a dose-dependent inhibitory effect on the release of NO in the cell supernatant. Among them， salvianolic acid B and ginsenoside Rb₁ had strong anti-inflammatory activity， with IC₅₀ values of （36.11±0.15） mg∙L^-1 and （27.24±0.54） mg∙L^-1， respectively. ConclusionThe quality evaluation method of Naomaili granules established in this study was accurate and reproducible. Four quality difference markers were screened out， and eight key pharmacodynamic substances of Naomaili granules against neuroinflammation were screened out by in vitro cell experiments.

Objective To explore the risk factors of painful diabetic neuropathy (PDN) in patients with type 2 diabetes mellitus (T2DM). Methods A total of 269 patients with type 2 diabetes mellitus (T2DM) who were treated in the Department of Endocrinology at Zhangjiagang Hospital Affiliated to Soochow University from January 2020 to December 2024 were selected. The patients were divided into two groups: T2DM without PDN (n=190) and T2DM with PDN (n=79). The general characteristics and biochemical indicators of the two groups of patients were compared. Multivariate logistic regression was used to analyze the associated factors with PDN in T2DM. The receiver operating characteristic (ROC) curve was used to evaluate fasting C-peptide (FC-P), body mass index (BMI), and disease duration to predict the risk of PDN. Results Compared with the T2DM group without concurrent PDN, the T2DM group with concurrent PDN had a longer disease course, lower BMI, higher HDL-C, lower FC-P, and a higher proportion of diabetic retinopathy. The differences between the two groups were statistically significant (P<0.05). The results of multivariate logistic regression analysis showed that BMI, duration, and FC-P were associated factors of PDN. Conclusion BMI, duration and FC-P are associated factors of painful neuropathy complicated with type 2 diabetes.

Objective:To establish the quality standard of Galla Turcica standard decoction; To provide a reference for the quality evaluation of its decoction granules.Methods:The moisture, total ash, acid-insoluble ash, and extract of Galla Turcica were assessed according to the guidelines specified in the Chinese Pharmacopoeia (Volume Ⅳ); high-performance liquid chromatography (HPLC) was employed to quantify the gallic acid content in sixteen batches of Galla Turcica and standard decoction, from which the transfer rate was calculated; the paste rate, moisture, and extract yield were quantitatively analyzed, establishing the comprehensive fingerprint of Galla Turcica standard decoction. The quantitative value transfer rules of common peaks from medicinal materials to standard decoction were studied through similarity evaluation and stoichiometric analysis. Results:The gallic acid content of the sixteen batches of Galla Turcica standard decoction ranged from 76.79 mg/g to 115.04 mg/g, the range of transfer rate was 24.02% to 47.82%, the extraction rate was 34.04% to 63.23%, the moisture was 1.11% to 2.06%, and the extract was 94.44% to 101.75%. A total of nine characteristic peaks were identified in the fingerprints of the sixteen batches of Galla Turcica and standard decoction, and four characteristic peaks were identified: gallic acid (GA), 1, 2, 3, 6 - tetragalloylglucose (TeGG), ellagic acid (EA) and 1, 2, 3, 4, 6, - pentagalloylglucose (PGG). Clustering analysis revealed that at a measurement distance of fifteen, the sixteen batches could be grouped into two categories. Principal component analysis (PCA) showed that three principal components were extracted with a cumulative variance contribution rate of 84.204%, which could represent most of the information of the samples.Conclusions:The standard decoction preparation process developed in this study adheres to the requirements of traditional decoction methods and has proven to be both effective and feasible. This method can be used for the research and quality evaluation of standard decoction of Galla Turcica, and provide a basis for subsequent formulation granules and related research.

With the continuous development of society, a large number of new chemicals are continuously emerging, which presents a challenge to current risk assessment and safety management of chemicals. Traditional toxicology research methods have certain limitations in quickly, efficiently, and accurately assessing the toxicity of many chemicals, and cannot meet the actual needs. In response to this challenge, computational toxicology that use mathematical and computer models to achieve the prediction of chemical toxicity has emerged. In the meantime, as researchers increasingly pay attention to understanding the interaction mechanisms between exogenous chemical substances and the body from the system level, and multiomics technologies develop rapidly such as genomics, transcriptomics, proteomics, and metabolomics, huge amounts of data have been generated, providing rich information resources for studying the interactions between chemical substances and biological molecules. System toxicology and network toxicology have also developed accordingly. Of these, network toxicology can integrate these multiomics data to construct biomolecular networks, and then quickly predict the key toxicological targets and pathways of chemicals at the molecular level. This paper outlined the concept and development of network toxicology, summarized the main methods and supporting tools of network toxicology research, expounded the application status of network toxicology in studying potential toxicity of exogenous chemicals such as agricultural chemicals, environmental pollutants, industrial chemicals, and foodborne chemicals, and analyzed the development prospects and limitations of network toxicology research. This paper aimed to provide a reference for the application of network toxicology in other fields.

Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

Grounded in the theory of "all marrows dominated by brain", this study explored the therapeutic mechanism of Zuogui Pills in modulating the oxytocin(OXT)/oxytocin receptor(OXTR) feed-forward loop in the treatment of postmenopausal osteoporosis(PMOP). A PMOP rat model was established using ovariectomy, and 70 Sprague-Dawley female rats were randomly divided into the following groups: sham operation group, model group, estradiol group(17β-estradiol, 0.05 mg·kg~(-1)·d~(-1)), Zuogui Pills low, medium, and high dose groups(0.2, 0.4, 0.8 g·kg~(-1)·d~(-1), respectively), and an antagonist group(atosiban 0.9 mg·kg~(-1)·d~(-1) + 17β-estradiol 0.05 mg·kg~(-1)·d~(-1) + Zuogui Pills 0.4 g·kg~(-1)·d~(-1)). After 12 weeks of model establishment, treatment was administered by gavage once daily for another 12 weeks, followed by sample collection. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of estrogen(E_2), OXT, tartrate-resistant acid phosphatase(TRACP-5b), and bone alkaline phosphatase(BALP). Histopathological changes in the left distal femur were observed through hematoxylin and eosin(HE) staining. Micro-computed tomography(micro-CT) was used to analyze the microstructure of the right distal femur. Western blot was employed to detect the expression levels of OXTR, small GTP-binding protein Ras, Raf1 proto-oncogene(Raf1), mitogen-activated protein kinase kinase 1/2(MEK1/2), and extracellular signal-regulated kinase 1/2(ERK1/2), and their phosphorylated forms in tibial tissues. Compared with the model group, the Zuogui Pills medium and high dose groups showed significantly increased levels of E_2, OXT, and BALP, with a notable decrease in TRACP-5b levels. Morphologically, the trabeculae in the left distal femur were more tightly arranged. The fibrous structure in the right distal femur was significantly improved in the Zuogui Pills high dose group. Additionally, the expression of OXTR, Ras, p-Raf1, p-MEK1/2, and p-ERK1/2 proteins in tibial tissues was significantly increased. The therapeutic effect of the Zuogui Pills high dose group was partially inhibited when an OXTR antagonist was administered. These findings suggest that Zuogui Pills can regulate the OXT/OXTR feed-forward loop, activate the phosphorylation of the downstream Ras/Raf1/MEK/ERK signaling pathway, and ultimately improve bone mineral density, thereby exerting therapeutic effects in PMOP.
Animals ; Rats, Sprague-Dawley ; Rats ; Female ; Drugs, Chinese Herbal/administration & dosage* ; Oxytocin/genetics* ; Receptors, Oxytocin/genetics* ; Humans ; Osteoporosis, Postmenopausal/genetics* ; Bone and Bones/drug effects* ; Brain/drug effects* ; Bone Marrow/drug effects*

As a high-quality moxibustion material, Artemisia stolonifera has high economic value and research prospects. However, due to difficulties in seed germination, its wild germplasm resources are sparsely distributed in China. This study used young stem segments grown in the current year to investigate the effects of explant sterilization, different combinations and concentrations of plant growth regulators on the proliferation and rooting of adventitious shoots, with the aim of constructing an in vitro rapid propagation technology system for A. stolonifera. The results showed that the lowest contamination rate of 25.83% was achieved when sterilizing the stem segments by rinsing with running water for 30 min, soaking in 75% ethanol for 30 s, followed by a 5 min treatment with 0.1% HgCl_2, 10 min with 8% NaClO, and 10 min with 0.6% phytosaniline. There was no browning of the stem segments, and surface sterilization of the A. stolonifera stem segments was successfully achieved. In the induction culture phase, when the concentration of kinetin(KT) was 0.05 mg·L~(-1) and 6-benzylaminopurine(6-BA) was 0.05 mg·L~(-1), the adventitious shoot proliferation coefficient was 2.02, effectively promoting the proliferation and growth of A. stolonifera. In the rooting culture phase, 0.1 mg·L~(-1) 1-naphthaleneacetic acid(NAA) effectively induced A. stolonifera test-tube seedlings to root within a short period, achieving a rooting rate of 100%. The addition of a small amount of activated charcoal also promoted rooting and strengthened seedling growth. The survival rate of A. stolonifera seedlings transplanted into a substrate consisting of 90% nutrient soil and 10% perlite was 100%. This study established an efficient in vitro rapid propagation system for A. stolonifera, overcoming difficulties with seed germination, shortening the breeding cycle, and reducing production and planting costs. It provides technical support for the introduction, domestication, seedling propagation, germplasm conservation, and industrial development of A. stolonifera.
Artemisia/drug effects* ; Tissue Culture Techniques/methods* ; Plant Growth Regulators/pharmacology* ; Plant Stems/drug effects* ; Plant Shoots/drug effects*

The medicinal materials of Bawei Chenxiang Pills(BCPs) were extracted via three methods: reflux extraction by water, reflux extraction by 70% ethanol, and extraction by pure water following reflux extraction by 70% ethanol, yielding three extracts of ST, CT, and CST. The efficacy of ST(760 mg·kg~(-1)), CT(620 mg·kg~(-1)), and CST(1 040 mg·kg~(-1)) were evaluated by acute myocardial ischemia(AMI) and p-chlorophenylalanine(PCPA)-induced insomnia in mice, respectively. Western blot was further utilized to investigate their hypnosis mechanisms. The main chemical components of different extracts were identified by the UPLC-Q-Exactive-MS technique. The results showed that CT and CST significantly increased the ejection fraction(EF) and fractional shortening(FS) of myocardial infarction mice, reduced left ventricular internal dimension at end-diastole(LVIDd) and left ventricular internal dimension at end-systole(LVIDs). In contrast, ST did not exhibit significant effects on these parameters. In the insomnia model, CT significantly reduced sleep latency and prolonged sleep duration, whereas ST only prolonged sleep duration without shortening sleep latency. CST showed no significant effects on either sleep latency or sleep duration. Additionally, both CT and ST upregulated glutamic acid decarboxylase 67(GAD67) protein expression in brain tissue. A total of 15 main chemical components were identified from CT, including 2-(2-phenylethyl) chromone and 6-methoxy-2-(2-phenylethyl) chromone. Six chemical components including chebulidic acid were identified from ST. The results suggested that chromones and terpenes were potential anti-myocardial ischemia drugs of BCPs, and tannin and phenolic acids were potential hypnosis drugs. This study enriches the pharmacological and chemical research of BCPs, providing a basis and reference for their secondary development, quality standard improvement, and clinical application.
Animals ; Drugs, Chinese Herbal/isolation & purification* ; Mice ; Male ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Humans ; Myocardial Infarction/drug therapy* ; Myocardial Ischemia/drug therapy*

This study aimed to investigate the effect and mechanism of Buyang Huanwu Decoction in regulating endoplasmic reticulum stress via the inositol-requiring enzyme 1α(IRE1α)/apoptosis signal-regulating kinase 1(ASK1)/c-Jun N-terminal kinase(JNK) pathway to improve neurological function in rats with cerebral ischemia/reperfusion injury(CIRI). SPF-grade male sprague-dawley(SD) rats were randomly divided into Sham group, model group, Buyang Huanwu Decoction group, and edaravone group. Except for the Sham group, the other groups were subjected to the modified suture method to establish a middle cerebral artery occlusion/reperfusion(MCAO/R) model. After treatment, neurological function was assessed using the Zea Longa scoring system. Gait analysis was used to detect the motor function. Detection of relative infarct area in brain tissue using 2,3,5-triphenyltetrazolium chloride(TTC) staining. Nissl staining was used to observe the structure of neuronal cells. Western blot and real-time fluorescence quantitative PCR(RT-qPCR) were used to detect IRE1α, ASK1, JNK, B cell lymphoma-2(Bcl-2), Bcl-2 related X protein(Bax), and Caspase-3 in the brain tissue. Immunohistochemistry was used to detect the positive expression of IRE1α, ASK1, and JNK. Immunofluorescence was used to detect the fluorescence expression levels of Bax, Bcl-2, and Caspase-3. The results showed that compared with the Sham group, the model group exhibited increased neurological scores(P<0.01), increased ratio of ground contact area and strength in both forelimbs(P<0.01), enlarged relative infarct area of brain tissue(P<0.05), and a reduced number of Nissl staining-positive cells(P<0.01). The protein and mRNA expression levels of IRE1α, ASK1, JNK, Bax, and Caspase-3 in brain tissue were significantly elevated, while those of Bcl-2 were decreased(P<0.05). Compared with the model group, both the Buyang Huanwu Decoction group and edaravone group showed reduced neurological scores(P<0.05), decreased ratio of ground contact area and strength in both forelimbs(P<0.05), smaller relative infarct area(P<0.05), alleviated neuronal damage, and increased number of Nissl staining-positive cells(P<0.05). The expression levels of IRE1α, ASK1, JNK, Bax, and Caspase-3 protein and mRNA in brain tissue were significantly reduced, while those of Bcl-2 were significantly increased(P<0.05). The results indicated that Buyang Huanwu Decoction can effectively improve brain injury in CIRI rats, and its mechanism of action may be related to regulating the endoplasmic reticulum stress IRE1α/ASK1/JNK signaling pathway.
Animals ; Male ; Rats, Sprague-Dawley ; Protein Serine-Threonine Kinases/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; MAP Kinase Kinase Kinase 5/genetics* ; Ischemic Stroke/physiopathology* ; Humans ; MAP Kinase Signaling System/drug effects* ; Apoptosis/drug effects* ; Endoribonucleases/genetics* ; JNK Mitogen-Activated Protein Kinases/genetics* ; Endoplasmic Reticulum Stress/drug effects* ; Multienzyme Complexes