Evaluation on the Potential for Hepatotoxic Components from Herba Epimedii to Induce Apoptosis in HepG2 Cells and the Analysis of the Influence of Metabolism in Liver Microsomes.

The potential hepatotoxicity of Herba Epimedii is a focal point in traditional Chinese medicine security applications. As determined in our previous study, the flavonoid constituents of Herba Epimedii, sagittatoside A, icariside I, baohuoside I and icaritin, are related to the hepatotoxicity of this herb. However, the hepatotoxic mechanism of these components needs to be clarified further, and whether these components can maintain their injury action following liver metabolism needs to be confirmed. Herein, the effects of sagittatoside A, icariside I, baohuoside I and icaritin on the apoptosis of HepG2 cells and the expression of key proteins, including Bax, Bcl-2, Caspase-3 and Caspase-9, were evaluated. Moreover, with liver microsome incubation, the influences of metabolism on the apoptotic activities of these components were investigated. Then, by HPLC-MS/MS analyses, the in vitro metabolic stability of these components was determined after incubation with different kinds of liver microsomes to explain the reason for the influence. The results suggested that sagittatoside A, baohuoside I and icaritin could induce apoptosis, which is likely to be closely related to the induction of the intrinsic apoptosis pathway. After metabolic incubation, the sagittatoside A and icaritin metabolism mixture could still induce apoptosis due to less metabolic elimination, while the icariside I and baohuoside I metabolism mixtures respectively got and lost the ability to induce apoptosis, probably due to quick metabolism and metabolic transformation. The findings of this study may provide important references to explore the material basis and mechanism of the hepatotoxicity of Herba Epimedii.

[Difference in liver injury induced by dictamnine between males and females: based on untargeted metabolomics].

In recent years, reports of adverse reactions related to traditional Chinese medicine(TCM) have been on the rise, especially some traditionally considered "non-toxic" TCM(such as Dictamni Cortex). This has aroused the concern of scholars. This study aims to explore the metabolomic mechanism underlying the difference in liver injury induced by dictamnine between males and females through the experiment on 4-week-old mice. The results showed that the serum biochemical indexes of liver function and organ coefficients were significantly increased by dictamnine(P<0.05), and hepatic alveolar steatosis was mainly observed in female mice. However, no histopathological changes were observed in the male mice. Furthermore, a total of 48 differential metabolites(such as tryptophan, corticosterone, and indole) related to the difference in liver injury between males and females were screened out by untargeted metabolomics and multivariate statistical analysis. According to the receiver operating characteristic(ROC) curve, 14 metabolites were highly correlated with the difference. Finally, pathway enrichment analysis indicated that disorders of metabolic pathways, such as tryptophan metabolism, steroid hormone biosynthesis, and ferroptosis(linoleic acid metabolism and arachidonic acid metabolism), may be the potential mechanism of the difference. Liver injury induced by dictamnine is significantly different between males and females, which may be caused by the disorders of tryptophan metabolism, steroid hormone biosynthesis, and ferroptosis pathways.

Psoraleae Fructus Ethanol Extract Induced Hepatotoxicity via Impaired Lipid Metabolism Caused by Disruption of Fatty Acid ß-Oxidation.

Herb-induced liver injury (HILI) is gradually increasing, and Psoraleae Fructus (PF) has been reported to induce hepatotoxicity. However, its underlying toxicity mechanism has been only poorly revealed. In this paper, we attempted to explore the liver injury and mechanism caused by Psoraleae Fructus ethanol extract (PFE). First, we administered PFE to mice for 4 weeks and evaluated their serum liver function indices. H&E staining was performed to observe the pathological changes of the livers. Oil red O staining was used to visualize hepatic lipids. Serum-untargeted metabolomics and liver proteomics were used to explore the mechanism of PF hepatotoxicity, and transmission electron microscopy was determined to assess mitochondria and western blot to determine potential target proteins expression. The results showed that PFE caused abnormal liver biochemical indicators and liver tissue injury in mice, and there was substantial fat accumulation in liver tissue in this group. Furthermore, metabolomic analysis showed that PFE changed bile acid synthesis, lipid metabolism, etc., and eight metabolites, including linoleic acid, which could be used as potential biomarkers of PFE hepatotoxicity. Proteomic analysis revealed that differential proteins were clustered in the mitochondrial transmembrane transport, the long-chain fatty acid metabolic process and purine ribonucleotide metabolic process. Multiomics analysis showed that eight pathways were enriched in both metabolomics and proteomics, such as bile secretion, unsaturated fatty acid biosynthesis, and linoleic acid metabolism. The downregulation of SLC27A5, CPT1A, NDUFB5, and COX6A1 and upregulation of cytochrome C and ABCC3 expressions also confirmed the impaired fatty acid oxidative catabolism. Altogether, this study revealed that PFE induced hepatotoxicity by damaging mitochondria, reducing fatty acid ß-oxidation levels, and inhibiting fatty acids ingested by bile acids.

[Correlation between drug-induced liver injury in rats caused by Xianling Gubao oral preparation and extraction process].

In light of the liver injury risk associated with the oral administration of Xianlin Gubao oral preparation, this study compared the differences in liver injury induced by two different extraction processes in rats and explored the correlation between hepatotoxicity and extraction process from the perspective of the differences in the content of the relevant components. Thirty male Sprague-Dawley(SD) rats were randomly divided into a normal group, tablet extract groups of different doses, and capsule extract groups of different doses, with 6 rats in each group. Each group received continuous oral administration for 4 weeks. The assessment of liver injury caused by different extracts was conducted by examining rat body weight, liver function blood biochemical indicators, liver coefficient, and liver pathological changes. In addition, a high-performance liquid chromatography(HPLC) method was established to simultaneously determine the content of icariin, baohuoside I, and bakuchiol in the extracts to compare the differences in the content of these three components under the two extraction processes. The results showed that both extracts caused liver injury in rats. Compared with the normal group, the tablet extract groups, at the studied dose, led to slow growth in body weight, a significant increase in triglyceride levels(P<0.05), a significant decrease in liver-to-brain ratio(P<0.05), and the appearance of hepatic steatosis. The capsule extract groups, at the studied dose, resulted in slow growth in body weight, a significant increase in aspartate aminotransferase levels(P<0.05), a significant decrease in body weight, liver weight, and liver-to-brain ratio(P<0.05), and the presence of hepatic steatosis and inflammatory cell infiltration. In comparison, the capsule extraction process had a higher risk of liver injury. Furthermore, based on the completion of the liquid chromatography method, the content of icariin and baohuoside Ⅰ in the capsule extract groups was 0.83 and 0.81 times that in the tablet extract groups, respectively, while the bakuchiol content in the capsule extract group was 29.80 times that in the tablet extract groups, suggesting that the higher risk of liver injury associated with the capsule extraction process may be due to its higher bakuchiol content. In summary, the differences in rat liver injury caused by the two extracts are closely related to the extraction process. This should be taken into consideration in the formulation production and clinical application.

Multi-Omics Integration to Reveal the Mechanism of Hepatotoxicity Induced by Dictamnine.

Herb-induced liver injury (HILI) has become a great concern worldwide due to the widespread usage of herbal products. Among these products is Dictamni Cortex (DC), a well-known Traditional Chinese Medicine (TCM), widely used to treat chronic dermatosis. Dictamni Cortex has drawn increasing attention because of its hepatotoxicity caused by the hepatotoxic component, dictamnine. However, the potential hepatotoxicity mechanism of dictamnine remains unclear. Therefore, this study aimed to use the multi-omics approach (transcriptomic, metabolomic, and proteomic analyses) to identify genes, metabolites, and proteins expressions associated with dictamnine-induced hepatotoxicity. A study on mice revealed that a high dose of dictamnine significantly increases serum aspartate aminotransferase (AST) activity, total bilirubin (TBIL), and direct bilirubin (DBIL) levels, the relative liver weight and liver/brain weight ratio in female mice (P < 0.05 and P < 0.01), compared to the normal control group. Liver histologic analysis further revealed a high dose of dictamnine on female mice caused hepatocyte vesicular steatosis characterized by hepatocyte microvesicles around the liver lobules. The expressed genes, proteins, and metabolites exhibited strong associations with lipid metabolism disorder and oxidative stress. Dictamnine caused increased oxidative stress and early hepatic apoptosis via up-regulation of glutathione S transferase a1 (GSTA1) and Bax/Bcl-2 ratio and down-regulation of the antioxidative enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase 1 (GPx-1). Besides, the up-regulation of Acyl-CoA synthetase long-chain family member 4 (ACSL4) and down-regulation of acetyl-coa acetyltransferase 1 (ACAT1) and fatty acid binding protein 1 (FABP-1) proteins were linked to lipid metabolism disorder. In summary, dictamnine induces dose-dependent hepatotoxicity in mice, which impairs lipid metabolism and aggravates oxidative stress.

Schisandra Inhibit Bleomycin-Induced Idiopathic Pulmonary Fibrosis in Rats via Suppressing M2 Macrophage Polarization.

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause and limited to the lungs. Schisandrae chinensis fructus (Wuweizi, Schisandra) is commonly used traditional Chinese medicines (TCM) for the treatment of pulmonary fibrosis, bronchitis, and other lung diseases in China. In this study, we investigated the therapeutic effect of Schisandra on IPF which is induced by bleomycin (BLM) in rats and the inhibition of alternatively activated macrophage (M2) polarization. Bleomycin-induced pulmonary fibrosis was used as a model for IPF, and rats were given drug interventions for 7 and 28 days to evaluate the role of Schisandra in the early oxidative phase and late fibrotic phases of BLM-induced pulmonary injury. The data showed that Schisandra exerted protective effects on BLM-induced pulmonary injury in two phases, which were improving inflammatory cell infiltration and severe damages of lung architectures and decreasing markers of M2 subtype. In order to prove the inhibitory effect of Schisandra on M2 polarization, in vitro experiments, we found that Schisandra downregulated the M2 ratio, which confirmed that the polarization of M2 was suppressed. Moreover, Schisandra blocked TGF-ß1 signaling in AMs by reducing the levels of Smad3 and Smad4; meanwhile, the upregulation of Smad7 by Schisandra also promoted the effect of inhibition on the TGF-ß1/Smad pathway. These results demonstrate that suppression of M2 polarization by Schisandra is associated with the development of IPF in rats.

[Study of macrophage polarization on pulmonary fibrosis and signaling pathway].

Macrophages are a group of immune cells with pluripotency and plasticity that can differentiate into different phenotypes under different microenvironments in vitro and in vivo. In the development of pulmonary fibrosis, there are alveolar macrophages and interstitial macrophages, which are polarized to different cell phenotypes at different stages of development. And their polarized phenotypes include M1 macrophages and M2 macrophages. In the inflammation early stages of pulmonary fibrosis, the increase of classical activated macrophages are helpful to clear pathogenic microorganisms and promote the progress of inflammation. In the fibrosis stage, the alternatively activated macrophages increased, which inhibiting the inflammatory reaction or directly promoting tissue fibrosis, on the other hand, it also promoting the fibrosis degradation. To clarify the polarization and polarization mechanisms of macrophages in pulmonary fibrosis will be conducive to the treatment of pulmonary fibrosis. In IPF, the polarization mechanism of M1 and M2 is closely related to TGF-ß1/Smad. TGF-ß1/Smad pathway plays an important regulatory role in liver fibrosis, renal fibrosis, myocardial fibrosis, scars, tumors and other diseases. Blocking the signaling of TGF-ß1 by Smad3 and Smad4 is beneficial to inhibit the polarization of AM, which in turn helps to inhibit the progression of IPF.

[Origin of lifting and lowering theory and its herb pair study].

Lifting and lowering theory is one of the important basis for guiding clinical medication. Through the study of ancient books and literature, we learned that lifting and lowering theory was originated in Huangdi Neijing, practiced more in the Shanghan Zabing Lun, established in Yixue Qiyuan, and developed in Compendium of Materia Medica and now. However, lifting and lowering theory is now mostly stagnated in the theoretical stage, with few experimental research. In the clinical study, the guiding role of lifting and lowering theory to prescriptions?mainly includes opposite?role?of lift and lower medicine property, mutual promotion of lift and lower medicine property, main role of lift medicine property and main role of lower medicine property. Under the guidance of lifting and lowering theory, the herb pair compatibility include herb combination of lift medicine property, herb combination of lift and lower medicine property and herb combination of lower medicine property. Modern biological technology was used in this study to carry out experimental research on the lifting and lowering theory, revealing the scientific connotation of it, which will help to promote clinical rational drug use.

[Analysis of historical origin and standardization system construction of Citri Grandis Exocarpium].

To understand the history development and changes of Citri Grandis Exocarpium and initially establish its standard system after exploring the historical origins and modern development of Citri Grandis Exocarpium. In CNKI, Wanfang database and other academic search platforms were searched for literature on Citri Grandis Exocarpium and Chinese medicine standard system； the books related to its modern cultivation techniques and processing technology were also accessed, and after combining with the actual situation analysis, the prospective design of the standard system of Citri Grandis Exocarpium was finally established with research conclusion. The earliest records of the Citri Grandis Exocarpium were documented in the Northern and Southern Dynasties, but its medicinal value was discovered in the Song Dynasty. Its drug use was developed on the basis of Jupi（orange peel） and Citri Exocarpium Rubrum. In 21st century, a number of large-scale, intensive Citri Grandis Exocarpium bases have been formed due to high price, good planting efficiency, and rapid growth of cultivation areas. The standard system includes the technical specifications of seed selection and seedling cultivation of Citri Grandis Exocarpium, technical norms of cultivation, technical specifications of fertilizing and weeding, technical specifications of irrigation and drainage, technical standard of pest and disease control, standard of medicinal materials grade, standard of processing technology of sliced pieces and the quality standard of slices, etc.

Effects of Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum on chemotactic migration of alveolar macrophages and inters regions macrophages in rats.

OBJECTIVE: To observe the effects of serum containing Mahuang (Herba Ephedra Sinica) or Wuweizi (Fructus Schisandrae Chinensis) on the migration of alveolar macrophages (AM) and interstitial macrophages (IM) from normal rats, and to analyze and compare the mechanisms leading to cell migration differences. METHODS: Rats were randomly divided into three groups: Mahuang (Herba Ephedra Sinica), Wuweizi (Fructus Schisandrae Chinensis), and blank serum. After treatment with the herbs, serum was extracted from the rats. AM and IM were isolated from normal rats and cultured. The effects of Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum on normal rat AM and IM chemotactic migration were determined by transwell assays. The CC chemokine receptor (CCR) 2, CCR5, voltage-gated Kvl. 3 K+ channel (Kv1. 3), and voltage-gated Kvl. 5 K+ channel (Kv1. 5) protein levels were analyzed by western blotting. RESULTS: The migration quantities of AM and IM in the Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum groups were significantly higher than those in the blank serum group (P < 0.01). Compared with the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group, the migration quantity of cultured rat AM in the Mahuang (Herba Ephedra Sinica) medicated serum group was significantly increased (P < 0.01). Meanwhile, compared with the Mahuang (Herba Ephedra Sinica) medicated serum group, the migration quantity of cultured rat IM in the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group was significantly increased (P < 0.01). CCR2, CCR5, Kv1. 3, and Kv1. 5 proteins were expressed on the AM cell surface, and showed significantly higher expression in the Mahuang (Herba Ephedra Sinica) medicated serum group compared with the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group. In contrast, CCR5, Kv1.3, and Kv1.5 proteins were expressed on the IM cell surface, and showed significantly higher expression in the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group compared with the Mahuang (Herba Ephedra Sinica) medicated serum group. CONCLUSION: Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) can promote AM and IM migration ability, with Mahuang (Herba Ephedra Sinica) targeting AM more apparently and Wuweizi (Fructus Schisandrae Chinensis) targeting IM more apparently. The mechanism may be that, by stimulating cells, Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) promote expression of CCR2 and CCR5 receptors on the AM and IM cell surface, which pass signals to Kvl.3 and Kvl.5 ion channels, leading to changes in the cytoskeleton, and ultimately promoting chemotactic cell migration.

[National physician master Jin Shiyuan's dispensing technology of Rehmanniae Radix Praeparata based on Li Shizhen's pharmaceutical academic thought].

To collect the historical origin, processing methods and clinical application of Rehmanniae Radix Preparata in Compendium of Materia Medica, compare and analyze the theoretical knowledge and relevant practical operation of national physician master Jin Shiyuan, which is beneficial for the inheritance and development of Rehmanniae Radix Preparata's clinical dispensing technology. In the analysis process, CNKI was searched with "Rehmanniae Radix Preparata", "Processing method", "Clinical application" "Li Shizhen", "Jin Shiyuan", and "Dispensing technology" as keywords. In addition, Shennong's Herbal Classic, Bencao Tujing (illustrated Classics of Materia Medica), Compendium of Materia Medica, Jingyue Quanshu (Jingyue's Complete Works) and related ancient books were accessed systematically to summarize the historical change of processing methods and efficacy of Rehmanniae Radix Praeparata. Professor Jin Shiyuan emphasizes the clinical dispensing technology of Rehmanniae Radix Praeparata, including its nature identification technology, clinical processing technology, prescription audit technology, prescription coping technology, drug delivery technology, clinical decocting technology, purchasing management technology as well as storage, maintenance and supply technology. Through the collation and research, it was confirmed that historical origin, processing methods and clinical application of Rehmanniae Radix Preparata were recorded in details in Compendium of Materia Medica. Steaming method of Rehmanniae Radix Preparata was originated from Synopsis of Golden Chamber. Li Shizhen attached great importance to the processing method of "steaming and drying alternatively for nine times" for Rehmanniae Radix Preparata, and differentiated it from Radix Rehmanniae Recen and fresh rehmannia root in clinical applications. Professor Jin Shiyuan has developed and improved the clinical dispensing technology of Rehmanniae Radix Praeparata, and carried forward the essence of Li Shizhen's pharmaceutical academic thought with his own proficient knowledge structure in medicine, providing scientific pharmaceutical service for clinical application of Rehmanniae Radix Preparata in future.

[National physician master Jin Shiyuan's dispensing technology of Aconiti Lateralis Radix Praeparata based on Li Shizhen's pharmaceutical academic thought].

To collect Li Shizhen's experience in Aconiti Lateralis Radix Praeparata identification and clinical application, compare and analyze national physician master Jin Shiyuan's practical operation and theoretical knowledge, which is beneficial for the inheritance and improvement of Aconiti Lateralis Radix Praeparata clinical dispensing technology. In the analysis process, CNKI, Wanfang and other databases were searched with "Aconiti Lateralis Radix Praeparata", "Li Shizhen", "pharmacological method state theory" "Jin Shiyuan" and "Chinese medicine dispensing technology" as the key words. In addition, Treatise on Febrile Disease, Compendium of Materia Medica, Chinese Pharmacopoeia(2015 edition), Notes to Medical Professions(Yi Zong Shuo Yue), and other medicine books were accessed to summarize the processing methods and decoction dosage of Aconiti Lateralis Radix Praeparata in both ancient and modern medicine, and in consideration of technical research and practice operation, Li Shizhen's description of Aconiti Lateralis Radix Praeparata and Professor Jin Shiyuan's research on Aconiti Lateralis Radix Praeparata dispensing technology were analyzed and collected. Li Shizhen recorded the nature identification and clinical application of Aconiti Lateralis Radix Praeparata by using pharmacological method state theory in Compendium of Materia Medica. National physician master Jin Shiyuan carries forward the essence of Li Shizhen's pharmaceutical academic thought with his own proficient knowledge structure in medicine, providing scientific pharmaceutical service for clinical application of Aconiti Lateralis Radix Praeparata Professor. Jin Shiyuan put forward the dispensing technology for the first time, including nature identification technology, clinical processing technology, clinical decocting technology, prescription coping technology, and class specifications of Aconiti Lateralis Radix Praeparata. In this paper, Aconiti Lateralis Radix Praeparata was used as an example to analyze the key dispensing technology of traditional Chinese medicine, and apply the key dispensing technology of traditional Chinese medicine in various commonly used Chinese medicines in the future.