In order to explore the possible role and molecular mechanism of the combined action of leech and bear bile in liver and gallbladder diseases, this study first used network pharmacology methods to screen the components and targets of leech and bear bile, as well as the related target genes of liver and gallbladder diseases. The selected key genes were subjected to interaction network and GO/KEGG enrichment analysis. Then, using sodium oleate induced HepG2 cell lipid deposition model and DL-ethionine induced mouse fatty liver model, the activity of leech and bear bile alone and in combination in reducing liver fat was evaluated in vitro and in vivo, and the expression of key pathway related proteins suggested by network pharmacology was detected by Western blot. The results of network pharmacology analysis showed that the active ingredients of leech and bear bile have 295 intersecting targets with liver and gallbladder related diseases, involving more than 200 signaling pathways, including the PI3K/Akt signaling pathway and phospholipase D signaling pathway closely related to glucose and lipid metabolism. The results of in vitro validation experiments showed that both leech and bear bile, alone and in combination, can significantly inhibit the lipid deposition induced by sodium oleate in human liver cells, reduce the triacylglycerol level in cell culture supernatant, and inhibit the lipid content in liver cells. The observation results of Nile red staining confocal microscopy showed that the combination of leech and bear bile had better activity in reducing lipid deposition in liver cells compared to using them alone. In a mouse fatty liver model, the combination of leech and bear bile can better reduce elevated organ indices, blood lipids, and liver lipid levels, as well as lower the levels of serum liver injury biomarkers. The animals used in this experiment and related disposal meet the requirements of animal welfare. Before the experiment, it was reviewed and approved by IACUC, Institute of Materia Medica, Chinese Academy of Medical Sciences. The Western blot experiment results showed that the combination of leech and bear bile can significantly upregulate the expression levels of p-PI3K and p-Akt proteins, and increase the p-PI3K/PI3K and p-Akt/Akt ratios, which is consistent with the predicted results of network pharmacology. The combination of leech and bear bile has great potential for treating fatty liver disease, and activating the PI3K/Akt pathway may be one of the important mechanisms for reducing lipid deposition in liver cells.

OBJECTIVE To evaluate the quality of evidence in the systematic evaluation/meta-analysis of traditional Chinese medicine （TCM） for diabetes retinopathy （DR） based on the GRADE system. METHODS Chinese and English databases were searched to obtain the relevant studies of systematic evaluation/meta-analysis of traditional Chinese medicine in the treatment of DR. The search time was from the establishment of each database to January 13th， 2024. According to the inclusion and exclusion criteria， literature screening was conducted. After extracting relevant information from the included literature， the GRADE system was used to evaluate the quality level of the evidence body in the included studies， and the evidence of the outcome indicators was integrated and summarized. RESULTS A total of 51 studies were ultimately included， encompassing 135 outcome indexes. Among these， 19 indicators （14.1%） were of high quality， 87 （64.4%） were of medium quality， 26 （19.3%） were of low quality， and 3 （2.2%） were of very low quality. Overall， the evidence quality of the outcome indicators in the included studies was medium to low quality. The integrated results of evidence on the efficacy of outcome indexes showed that compared with conventional Western medicine， calcium dobesilate or placebo， TCM had significant advantages in improving overall efficacy， reducing bleeding spot area， reducing macular foveal thickness， and increasing visual improvement rate. In addition，the combination of TCM and conventional Western medicine or calcium dobesilate was significantly more effective than using conventional Western medicine or calcium dobesilate alone. CONCLUSIONS The overall quality of the evidence in the systematic evaluation/meta-analysis study on the treatment of DR with TCM is medium to low quality. Based on existing research findings， TCM demonstrates good clinical efficacy in the treatment of DR.

ObjectiveTo investigate the effect of Liuwei Dihuangwan drug-containing serum （LDP） on epithelial-mesenchymal transition （EMT） and the expression of cancer stem cell （CSC） properties in 4T1 cells from triple-negative breast cancer by intervening myeloid-derived suppressor cells （MDSCs）. MethodsSPF-grade female SD rats were randomly divided into three groups， which were given 0.39， 1.94， 3.89 g·kg^-1·d^-1 suspension of Liuwei Dihuangwan for 7 days， respectively， to prepare low-， medium-， and high-dose LDPs. 4T1 cells were inoculated subcutaneously into the mammary glands of SPF-grade female Balb/c mice to construct a transplantation tumor model. Bone marrow cells were extracted from the tibia and femur and induced into MDSCs in vitro. The cell counting kit-8 （CCK-8） assay was used to detect the viability of 4T1 cells and MDSCs. The number of MDSCs and the expressions of CSC surface markers CD44 and CD24 in 4T1 cells were detected by flow cytometry （FC）. The migration， invasion， and proliferation of 4T1 cells were detected by cell scratch assay， Transwell invasion assay， and plate colony-forming assay， respectively. Western blot （WB） was used to detect the protein expression of transforming growth factor-β （TGF-β）， nuclear factor-κB （NF-κB）， C-X-C motif chemokine ligand 2 （CXCL2）， E-cadherin， and N-cadherin. The expression of EMT-related proteins E-cadherin and N-cadherin were detected by immunofluorescence （IF）. ResultsCompared with the normal group， LDP showed no significant inhibitory effect on the cell viability of 4T1 cells， but it significantly reduced the viability and number of MDSCs and reduced the number of MDSCs， as well as the expression of TGF-β （P<0.05， P<0.01）. The migration， invasion， and proliferation of 4T1 cells were increased after co-culture with MDSCs （P<0.05， P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin and the proportion of CSC （CD44⁺CD24^-） were elevated （P<0.05， P<0.01）， while the expression of E-cadherin was decreased （P<0.05）. After the intervention of MDSCs with LDP， followed by co-culture with 4T1 cells， the migration， invasion， and proliferation of 4T1 cells were obviously reduced （P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin were decreased （P<0.05， P<0.01）， and the expression of E-cadherin was increased （P<0.05， P<0.01）. There was no statistical difference in the proportion of CSC （CD44⁺CD24^-） in 4T1 cells. However， the proportion of CSC （CD44⁺CD24^-） was decreased in the co-culture system of 4T1 cells and MDSCs with LDP intervention （P<0.05， P<0.01）. ConclusionLDP can reduce the viability and number of MDSCs and the expression of TGF-β， NF-κB， and CXCL2， reverse EMT， and reduce the characteristic expression of CSC to inhibit the migration， invasion， and proliferation of 4T1 cells.

ObjectiveTo investigate the effect of Liuwei Dihuangwan drug-containing serum （LDP） on epithelial-mesenchymal transition （EMT） and the expression of cancer stem cell （CSC） properties in 4T1 cells from triple-negative breast cancer by intervening myeloid-derived suppressor cells （MDSCs）. MethodsSPF-grade female SD rats were randomly divided into three groups， which were given 0.39， 1.94， 3.89 g·kg^-1·d^-1 suspension of Liuwei Dihuangwan for 7 days， respectively， to prepare low-， medium-， and high-dose LDPs. 4T1 cells were inoculated subcutaneously into the mammary glands of SPF-grade female Balb/c mice to construct a transplantation tumor model. Bone marrow cells were extracted from the tibia and femur and induced into MDSCs in vitro. The cell counting kit-8 （CCK-8） assay was used to detect the viability of 4T1 cells and MDSCs. The number of MDSCs and the expressions of CSC surface markers CD44 and CD24 in 4T1 cells were detected by flow cytometry （FC）. The migration， invasion， and proliferation of 4T1 cells were detected by cell scratch assay， Transwell invasion assay， and plate colony-forming assay， respectively. Western blot （WB） was used to detect the protein expression of transforming growth factor-β （TGF-β）， nuclear factor-κB （NF-κB）， C-X-C motif chemokine ligand 2 （CXCL2）， E-cadherin， and N-cadherin. The expression of EMT-related proteins E-cadherin and N-cadherin were detected by immunofluorescence （IF）. ResultsCompared with the normal group， LDP showed no significant inhibitory effect on the cell viability of 4T1 cells， but it significantly reduced the viability and number of MDSCs and reduced the number of MDSCs， as well as the expression of TGF-β （P<0.05， P<0.01）. The migration， invasion， and proliferation of 4T1 cells were increased after co-culture with MDSCs （P<0.05， P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin and the proportion of CSC （CD44⁺CD24^-） were elevated （P<0.05， P<0.01）， while the expression of E-cadherin was decreased （P<0.05）. After the intervention of MDSCs with LDP， followed by co-culture with 4T1 cells， the migration， invasion， and proliferation of 4T1 cells were obviously reduced （P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin were decreased （P<0.05， P<0.01）， and the expression of E-cadherin was increased （P<0.05， P<0.01）. There was no statistical difference in the proportion of CSC （CD44⁺CD24^-） in 4T1 cells. However， the proportion of CSC （CD44⁺CD24^-） was decreased in the co-culture system of 4T1 cells and MDSCs with LDP intervention （P<0.05， P<0.01）. ConclusionLDP can reduce the viability and number of MDSCs and the expression of TGF-β， NF-κB， and CXCL2， reverse EMT， and reduce the characteristic expression of CSC to inhibit the migration， invasion， and proliferation of 4T1 cells.

This study primarily investigates the effect of Liuwei Dihuang Pills on the activation and proliferation of female germline stem cells(FGSCs) in the ovaries and cortex of mice with premature ovarian failure(POF), and how it improves ovarian function. ICR mice were randomly divided into the control group, model group, Liuwei Dihuang Pills group, Liuwei Dihuang Pills double-dose group, and estradiol valerate group. A mouse model of POF was established by intraperitoneal injection of cyclophosphamide. After successful modeling, the mice were treated with Liuwei Dihuang Pills or estradiol valerate for 28 days. Vaginal smears were prepared to observe the estrous cycle and body weight. After the last administration, mice were sacrificed and sampled. Serum levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-Müllerian hormone(AMH) were measured by enzyme-linked immunosorbent assay(ELISA). Hematoxylin-eosin(HE) staining was used to observe ovarian morphology and to count follicles at all stages to evaluate ovarian function. Immunohistochemistry was used to detect the expression of mouse vasa homolog(MVH), a marker of ovarian FGSCs. Immunofluorescence staining, using co-labeling of MVH and proliferating cell nuclear antigen(PCNA), was used to detect the expression and localization of specific markers of FGSCs. Western blot was employed to assess the protein expression of MVH, octamer-binding transcription factor 4(Oct4), and PCNA in the ovaries. The results showed that compared with the control group, the model group exhibited disordered estrous cycles, decreased ovarian index, increased atretic follicles, and a reduced number of follicles at all stages. FSH and LH levels were significantly elevated, while AMH and E_2 levels were significantly reduced, indicating the success of the model. After treatment with Liuwei Dihuang Pills or estradiol valerate, hormone levels improved, the number of atretic follicles decreased, and the number of follicles at all stages increased. MVH marker protein and PCNA proliferative protein expression in ovarian tissue also increased. These results suggest that Liuwei Dihuang Pills regulate estrous cycles and hormone disorders in POF mice, promote the proliferation of FGSCs, improve follicular development in POF mice, and enhance ovarian function.
Animals ; Female ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Cell Proliferation/drug effects* ; Mice, Inbred ICR ; Ovary/cytology* ; Primary Ovarian Insufficiency/genetics* ; Follicle Stimulating Hormone/metabolism* ; Humans ; Anti-Mullerian Hormone/blood* ; Antineoplastic Agents/adverse effects* ; Luteinizing Hormone/metabolism* ; Cyclophosphamide/adverse effects*

The antidepressant activity and molecular mechanisms of Yueju Wan volatile oil were investigated. The Yueju Wan volatile oil was extracted by using supercritical CO_2. Gas chromatography-mass spectrometry(GC-MS) combined with network pharmacology identified 28 chemical constituents in Yueju Wan volatile oil, primarily terpenes and lactones. A total of 123 overlapping targets were associated with depression, including core targets of interleukin-1β(IL-1β), signal transducer and activator of transcription 3(STAT3), and caspase-3(CASP3). These targets were mainly involved in the prolactin, advanced glycation end products/receptor(AGE/RAGE), and phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) signaling pathways. A reserpine-induced depression mouse model was established to evaluate the therapeutic effects and mechanisms of Yueju Wan volatile oil. The effects of Yueju Wan volatile oil on depression-like behavior in mice were evaluated by analyzing body mass, body temperature index, tail suspension immobility time, forced swimming immobility time, and sucrose preference. Hematoxylin-eosin(HE) staining revealed neuronal protection of Yueju Wan volatile oil in the brain of mice. Enzyme-linked immunosorbent assay(ELISA) and Western blot were employed to detect the protein expression of AGEs, IL-1β, phosphorylated PI3K(p-PI3K), Akt, phosphorylated Akt(p-Akt), nuclear factor κB(NF-κB), and brain-derived neurotrophic factor(BDNF). Behavioral evaluation showed that Yueju Wan volatile oil could effectively control the decline of body mass and body temperature of depressed mice, reduce tail suspension and swimming immobility time, and enhance their preference for sucrose. Histopathological examination showed that Yueju Wan volatile oil could alleviate the neuronal damage in CA1 and dentate gyrus(DG) of the hippocampus of mice. ELISA and Western blot results showed that Yueju Wan volatile oil could significantly increase the protein expression levels of PI3K, Akt, and BDNF and significantly decrease the protein expression levels of AGEs, IL-1β, p-PI3K, p-Akt, and NF-κB in the hippocampus of mice. Furthermore, the p-PI3K/PI3K and p-Akt/Akt ratios were significantly decreased at medium and high doses. These findings suggest that the aromatherapy of Yueju Wan volatile oil can significantly improve reserpine-induced depression-like behavior in mice, which may be related to reducing the expression of neuronal membrane protein AGEs, reducing the phosphorylation levels of PI3K and Akt, inhibiting NF-κB entry into the nucleus, and alleviating the release of pro-inflammatory factors and nerve injury.
Animals ; Antidepressive Agents/chemistry* ; Mice ; Proto-Oncogene Proteins c-akt/immunology* ; Phosphatidylinositol 3-Kinases/immunology* ; Oils, Volatile/chemistry* ; Male ; Drugs, Chinese Herbal/chemistry* ; Signal Transduction/drug effects* ; Depression/metabolism* ; Glycation End Products, Advanced/immunology* ; Humans

Climate and land use changes may significantly impact the habitat distribution of Gastrodia elata, an endangered traditional medicinal plant. Accurately predicting its future potential suitable habitats is crucial for its conservation and sustainable development. This study integrates current distribution data of G. elata with 56 environmental variables and uses the MaxEnt model to predict changes in its suitable habitats under current climate conditions and four future climate scenarios(SSP1-2.6, SSP2-4.5, SSP3-7.0, and SSP5-8.5). The results show that October precipitation and December minimum temperature are key environmental factors influencing its distribution. Under the current climate, optimal habitats for G. elata are concentrated in montane forest areas in Sichuan, Yunnan, Guizhou, and Hubei, which meet the species' requirements for understory growth. Across all future scenarios, the suitable habitat of G. elata consistently shows a stable northward shift, with a steady increase in suitable areas, extending to the middle and lower reaches of the Yangtze River and the Huang-Huai region, and even expanding into Liaoning, Jilin, and southern Heilongjiang. Land use analysis, taking into account the protection of arable land and the utilization of forest resources, indicates that by 2100, under future climate conditions, arable land in medium-to high-suitability areas is expected to increase by 30%-124%. While the conversion of non-suitable forest land into suitable habitats is projected to increase by 5%-52%, the growth of medium-to high-suitability areas within forests is relatively modest, ranging from 1% to 24%. These findings highlight the need to balance agricultural expansion with forest resource conservation to ensure the long-term sustainability of G. elata and provide scientific guidance for future suitable habitat management.
Ecosystem ; China ; Climate Change ; Gastrodia/growth & development* ; Conservation of Natural Resources ; Plants, Medicinal/growth & development*

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

ObjectiveThis study explores the methods of lung tissue extraction and fixation required for pathological studies of fetal rats, based on the unique physiological structure of fetal rat lung tissue and existing lung tissue fixation techniques for adult rats. MethodsSix pregnant adult SD rats at 20.5 days of gestation were subjected to cesarean section to obtain fetal rats. Four healthy fetal rats with similar body weight, vital signs, and respiratory status were selected from each pregnant rat, and they were randomly divided into the following groups using a random number table: direct lung infiltration group, lung infiltration group after intratracheal infusion, whole-body infiltration group of fetal rats, and whole-body infiltration group after intratracheal infusion of fetal rats. To systematically compare and analyze the anatomical morphology under different fixation methods, lung tissues from four groups of fetal rats were harvested, perfused, and fixed, and the gross morphology of lung tissues in each group was observed. Paraffin sections were prepared and stained with Hematoxylin-Eosin (H&E). The histological morphology of the whole lung, alveoli, and bronchi was further examined under optical microscopy. ResultsIn the direct lung infiltration group, the hilar structures were unclear, lung lobation was indistinct, the shape was irregular, lung cavities were small, and alveoli and bronchi were shrunken. In the lung infiltration group after intratracheal infusion, the hilar structures were clear, lobation was pronounced, the shape was regular, lung cavities were large, and alveoli and bronchi were full. Both the whole-body infiltration group and whole-body infiltration group after intratracheal infusion of fetal rats exhibited visible lungs, hearts, skins, and other organs. The lung tissues of both groups showed obvious lobulation, irregular shape, and damage at the margins of lung lobes. In the whole-body infiltration group, the thoracic cavities of the fetus were flattened, lung cavities were small, and alveoli and bronchi were shrunken. In the whole-body infiltration group after intratracheal infusion of fetal rats, the fetal thoracic cavities were full, lung cavities were large, and alveoli and bronchi were relatively full. ConclusionThe lung infiltration after intratracheal infusion method for fetal rat lung tissue fixation outperforms direct lung infiltration, whole-body infiltration of fetal rats, and whole-body infiltration after intratracheal infusion of fetal rats in terms of preservation of the lung tissue's original morphology, paraffin sectioning, staining, and pathological observation and analysis. The embedding, sectioning, and staining processes are also simple and save consumables. Therefore, intratracheal infusion followed by lung infiltration method is recommended for fixation in histopathological observation of fetal rat lung tissue.

Objective To explore the mediating role of occupational well-being in the relationship between professional identity and safety behavior among nurses. Methods A total of 1 006 nurses from ten tertiary general hospitals in eight provincial administrative regions were selected as the research subjects using convenient sampling method. Their safety behavior, professional identity and occupational well-being were investigated using Nurse Safety Behavior Scale, Nurse Professional Identity Scale and Occupational Well-being Scale. Structural equation modeling was performed using AMOS 26.0 to examine the mediating effect of occupational well-being in the relationship between professional identity and safety behavior among nurses. Results The scores for safety behavior, professional identity, and occupational well-being were (53.0±6.1), (123.7±21.2) and (90.8±13.1), respectively. Safety behavior was positively correlated with both professional identity and occupational well-being (correlation coefficients were 0.50 and 0.50, respectively, both P<0.01). Professional identity was positively correlated with occupational well-being (correlation coefficient was 0.51, P<0.01). The multiple linear regression analysis results showed that the higher the professional identity and occupational well-being of nurses, the higher the level of safety behavior (both P<0.05). The result of mediating effect shows that the total effect of occupational identity on safety behavior was 0.498 [95% confidence interval (CI) was 0.405-0.576], and occupational well-being played a mediating role between professional identity and safety behavior among nurses with the mediation effect of 0.156 (95%CI was 0.112-0.205), accounting for 31.33% of the total effect. Conclusion The safety behavior of nurses is at a moderate level. Both professional identity and occupational well-being can affect the safety behavior of nurses. Professional identity can increase the safety behavior of nurses by affecting occupational well-being.