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

OBJECTIVE: To explore the displacement and pressure distribution of American Chiropractic in a model of third lumbar syndrome based on finite element analysis. METHODS: On March 2021, CT and MRI images of a 23-year-old male patient with right third lumbar syndrome were selected. A 3D stl model was established using Mimics and CATIA, and the data was imported into Hypermesh, Abaqus & ANSYS. The elastic modulus and Poisson's ratio of the affected side material were adjusted to establish its finite element model. Based on the comparison of the operating positions and routines of the American Chiropractic and the lumbar spine oblique pull method, but with differences in the focus and direction of force, the experimental group simulated the American Chiropractic with the healthy side (left side) lying position of the model. The upper endplate of L₃ and the lower part below L₃ twisted accordingly with the body position, we applied a vertical forward thrust of 246 N to the plane formed by the L₄, L₅ spinous processes and L₄ upper articular processes;The control group simulates the oblique pull method of the lumbar spine, requiring the model to lie on the healthy side (left side), fix the upper endplate of L₄, and perform a horizontal rotation along the longitudinal axis of L₃ vertebral body. At this time, the contact force in the upward direction is also set to 246 N. Compare the displacement and stress differences between the L₁-L₅ intervertebral bodies, intervertebral discs, articular processes, and transverse process muscles in two intervention models. RESULTS: ① Under safe load conditions, a test force of 246 N was applied to the model, and the maximum vertebral displacement occurred on the right side of the L₃ vertebral body (1.197 mm) after manual intervention in the control group. The vertebral displacement between L₁-L₅ induced by manual intervention in the experimental group was smaller than that of the control group's manual intervention (P<0.05). ② The maximum vertebral body stress occurred on the right side of the L₃ vertebral body after manual intervention in the control group (98.425 MPa). The stress on each vertebral body formed by the experimental group's manual intervention was lower than that of the control group's manual intervention (P<0.05). ③The maximum intervertebral disc stress occurred on the right side of the L_2,3 intervertebral disc (6.282 MPa) after manual intervention in the control group. ④ The maximum joint process stress occurred on the right side of the L₄ upper joint process after manual intervention in the experimental group (1.587 MPa). The joint process stress on the left side below L₁ and the left side above and below L₂ induced by manual intervention in the experimental group was lower than that of the control group (P<0.05). ⑤The maximum stress on the intertransverse process muscle was observed at the right lateral L₃ process end (31.960 MPa) of L_3,4 in the control group after manual intervention. The stress on the L_2,3 and L_4,5 segments of the intertransverse process muscle induced by manual intervention in the experimental group was lower than that of the control group's manual intervention (P<0.05). CONCLUSION The mechanical feedback of the L₁-L₅ vertebral body, the lower left side of the articular process L₁, the upper and lower left side of the articular process L₂, and the L_2,3 and L_4,5 segments of the transverse process muscle in the model indicates that performing American Chiropractic for the treatment of third lumbar transverse process syndrome can accurately hit the target pain point and allow the patient's tissue to form a low stress and low tension state after manual operation, thereby reducing the possibility of tissue damage caused by hypertonia after intervertebral joint movement, making it relatively safe. The application of American Chiropractic will be a new supplement to the traditional treatment plan for third lumbar transverse process syndrome.
Humans ; Finite Element Analysis ; Male ; Lumbar Vertebrae/physiopathology* ; Biomechanical Phenomena ; Young Adult ; Manipulation, Chiropractic ; Adult ; Tomography, X-Ray Computed ; Magnetic Resonance Imaging

OBJECTIVE: To illustrate the role of dehydrocorydaline (DHC) in chronic constriction injury (CCI)-induced neuropathic pain and the underlying mechanism. METHODS: C57BL/6J mice were randomly divided into 3 groups by using a random number table, including sham group (sham operation), CCI group [intrathecal injection of 10% dimethyl sulfoxide (DMSO)], and CCI+DHC group (intrathecal injection of DHC), 8 mice in each group. A CCI mouse model was conducted to induce neuropathic pain through ligating the right common sciatic nerve. On day 14 after CCI modeling or sham operation, mice were intrathecal injected with 5 µL of 10% DMSO or 10 mg/kg DHC (5 µL) into the 5th to 6th lumbar intervertebral space (L5-L6). Pregnant ICR mice were sacrificed for isolating primary spinal neurons on day 14 of embryo development for in vitro experiment. Pain behaviors were evaluated by measuring the paw withdrawal mechanical threshold (PWMT) of mice. Immunofluorescence was used to observe the activation of astrocytes and microglia in mouse spinal cord. Protein expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), phosphorylation of N-methyl-D-aspartate receptor subunit 2B (p-NR2B), and NR2B in the spinal cord or primary spinal neurons were detected by Western blot. RESULTS: In CCI-induced neuropathic pain model, mice presented significantly decreased PWMT, activation of glial cells, overexpressions of iNOS, TNF-α, IL-6, and higher p-NR2B/NR2B ratio in the spinal cord (P<0.05 or P<0.01), which were all reversed by a single intrathecal injection of DHC (P<0.05 or P<0.01). The p-NR2B/NR2B ratio in primary spinal neurons were also inhibited after DHC treatment (P<0.05). CONCLUSION An intrathecal injection of DHC relieved CCI-induced neuropathic pain in mice by inhibiting the neuroinflammation and neuron hyperactivity.
Animals ; Neuralgia/etiology* ; Mice, Inbred C57BL ; Analgesics/pharmacology* ; Neuroinflammatory Diseases/pathology* ; Constriction ; Male ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Mice, Inbred ICR ; Microglia/pathology* ; Spinal Cord/drug effects* ; Female ; Mice ; Tumor Necrosis Factor-alpha/metabolism* ; Disease Models, Animal ; Constriction, Pathologic/complications* ; Interleukin-6/metabolism* ; Astrocytes/metabolism* ; Chronic Disease ; Neurons/metabolism*

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets

Hepatic stellate cells (HSCs) are the primary fibrogenic cells in the liver, and their activation plays a crucial role in the development and progression of hepatic fibrosis. Here, we report that retinoid X receptor-alpha (RXRα), a unique member of the nuclear receptor superfamily, is a key modulator of HSC activation and liver fibrosis. RXRα exerts its effects by modulating calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-mediated activation of AMP-activated protein kinase-alpha (AMPKα). In addition, we demonstrate that K-80003, which binds RXRα by a unique mechanism, effectively suppresses HSC activation, proliferation, and migration, thereby inhibiting liver fibrosis in the CCl₄ and amylin liver NASH (AMLN) diet animal models. The effect is mediated by AMPKα activation, promoting mitophagy in HSCs. Mechanistically, K-80003 activates AMPKα by inducing RXRα to form condensates with CaMKKβ and AMPKα via a two-phase process. The formation of RXRα condensates is driven by its N-terminal intrinsic disorder region and requires phosphorylation by CaMKKβ. Our results reveal a crucial role of RXRα in liver fibrosis regulation through modulating mitochondrial activities in HSCs. Furthermore, they suggest that K-80003 and related RXRα modulators hold promise as therapeutic agents for fibrosis-related diseases.

Dysfunction of drug transporters significantly affects therapeutic outcomes and drug efficacy in patients with liver injury. Clinical and experimental evidence demonstrates that liver injury involves complex inter-organ interactions among the brain, eye, liver, intestine, and kidney. Recent advances in basic and clinical research have illuminated the physiologic and molecular mechanisms underlying transporter alterations in liver injury, particularly those associated with bilirubin, reactive oxygen species, ammonia, bile acid, and inflammatory factors. Notably, the influence of these transporter modifications on drug pharmacokinetics in liver injury patients remains inadequately understood. Additional research is necessary to fully comprehend these effects and their therapeutic implications. The documented alterations of transporters in distant organs across various liver diseases indicate that dosage modifications may be required when administering transporter-substrate drugs, including both traditional Chinese and Western medicines, to patients with liver dysfunction. This strategy helps maintain drug concentrations within therapeutic ranges while reducing adverse reactions. Furthermore, when utilizing transporter inducers or inhibitors clinically, consideration of their long-term effects on transporters and subsequent therapeutic impact is essential. Careful attention must be paid to avoid compromising the elimination of toxic metabolites and proteins when inhibiting these transporters. Similarly, prudent use of inducers or inducer-type therapeutic drugs is necessary to prevent enhanced drug resistance. This review examines recent clinical and experimental findings regarding the inter-organ interaction of drug transporters in liver injury conditions and their clinical relevance.
Humans ; Liver/drug effects* ; Animals ; Chemical and Drug Induced Liver Injury/metabolism* ; Membrane Transport Proteins/metabolism* ; Biological Transport ; Liver Diseases/drug therapy* ; Pharmaceutical Preparations/metabolism*

Objective To compare the pharmacokinetic profiles of the two teriflunomide tablets in healthy Chinese subjects under fasting and fed conditions and to evaluate their bioequivalence and safety.Methods A randomized,open,single-dose,parallel trial design was used to enroll 31 and 32 healthy Chinese male subjects in the fasting and fed groups,who were randomized to a single oral dose of 14 mg of either reference or test preparation of teriflunomide tablets.The plasma concentrations of teriflunomide were determined using liquid chromatography-tandem mass spectrometry method,and Phoenix WinNonlin 8.1 software was used to calculate pharmacokinetic parameters and perform bioequivalence analysis.Results Subjects received a single oral dose of the reference and test formulations of teriflunomide.The main pharmacokinetic parameters of teriflunomide in the fasting group were as follows:Cmax were(2.14±0.27)and(2.27±0.33)μg·mL-1,AUC0-72h were(105.70±11.20)and(107.72±11.77)μg·mL-1·h,tmax was 1.49 and 0.99 h;the main pharmacokinetic parameters of teriflunomide in the fed group were as follows:Cmaxwere(1.83±0.17)and(1.75±0.22)μg·mL-1,AUC0-72h were(102.66±9.18)and(101.57±13.01)μg·mL-1·h,tmax was 4.01 and 4.99 h.The 90％confidence intervals for the geometric means of Cmax and AUC0-72h for reference and test preparations in the fasting and fed groups were in the range of 80％to 125％.Conclusion The pharmacokinetic characteristics of the 2 formulations were similar under fasting and fed administration conditions,with good bioequivalence and safety;Postprandial administration may delay the time to peak of the drug.

AIM:To observe how total flavonoids of Pterocarya hupehensis Skan(PHSTF)affects the migra-tion and invasion of human rheumatoid fibroblast-like synoviocyte line MH7A.METHODS:The MH7A cells were divided into control group(without any treatment),low-,medium-and high-dose(6.25,12.5 and 25 mg/L,respectively)PHSTF groups,phosphatidylinositol 3-kinase(PI3K)inhibitor 740Y-P(10 μmol/L)group,and 740Y-P(10 μmol/L)+high-dose(25 mg/L)PHSTF group.The viability of the MH7A cells was determined by CCK-8 assay,while the migration and inva-sion were assessed by scratch and Transwell assays.The protein levels of matrix metalloproteinase-2(MMP-2),MMP-9,PI3K,p-PI3K,AKT and p-AKT were detected by Western blot.RESULTS:Compared with control group,the cell via-bility in PHSTF treatment groups was reduced(P<0.05),the cell wound healing area was significantly decreased(P<0.01),migratory and invasive cells in the lower chamber were significantly reduced(P<0.01),and the protein expres-sion of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT were decreased(P<0.01).Compared with high-dose PHSTF group,the addition of PI3K/AKT pathway agonist 740Y-P significantly increased the migration and invasion ability of MH7A cells(P<0.01)and elevated the protein expression of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT(P<0.01)under the treatment with PHSTF.CONCLUSION:Total flavonoids of Pterocarya hupehensis Skan could inhibit the migration and invasion of MH7A cells by regulating the PI3K/AKT signaling pathway.

Objective:To analyze the factors affecting delayed chemotherapy in children with Burkitt lymphoma (BL) and their influence on prognosis.Methods:Retrospective cohort study. Clinical data of 591 children aged ≤18 years with BL from May 2017 to December 2022 in China Net Childhood Lymphoma (CNCL) was collected. The patients were treated according to the protocol CNCL-BL-2017. According to the clinical characteristics, therapeutic regimen was divided into group A, group B and group C .Based on whether the total chemotherapy time was delayed, patients were divided into two groups: the delayed chemotherapy group and the non-delayed chemotherapy group. Based on the total delayed time of chemotherapy, patients in group C were divided into non-delayed chemotherapy group, 1-7 days delayed group and more than 7 days delayed group. Relationships between delayed chemotherapy and gender, age, tumor lysis syndrome before chemotherapy, bone marrow involvement, disease group (B/C group), serum lactate dehydrogenase (LDH) > 4 times than normal, grade Ⅲ-Ⅳ myelosuppression after chemotherapy, minimal residual disease in the interim assessment, and severe infection (including severe pneumonia, sepsis, meningitis, chickenpox, etc.) were analyzed. Logistic analysis was used to identify the relevant factors. Kaplan-Meier method was used to analyze the patients' survival information. Log-Rank was used for comparison between groups.Results:Among 591 patients, 504 were males and 87 were females, the follow-up time was 34.8 (18.6,50.1) months. The 3-year overall survival (OS) rate was (92.5±1.1)%,and the 3-year event-free survival (EFS) rate was (90.5±1.2)%. Seventy-three (12.4%) patients were in delayed chemotherapy group and 518 (87.6%) patients were in non-delayed chemotherapy group. The reasons for chemotherapy delay included 72 cases (98.6%) of severe infection, 65 cases (89.0%) of bone marrow suppression, 35 cases (47.9%) of organ dysfunction, 22 cases (30.1%) of tumor lysis syndrome,etc. There were 7 cases of chemotherapy delay in group B, which were seen in COPADM (vincristine+cyclophosphamide+prednisone+daunorubicin+methotrexate+intrathecal injection,4 cases) and CYM (methotrexate+cytarabine+intrathecal injection,3 cases) stages. There were 66 cases of chemotherapy delay in group C, which were common in COPADM (28 cases) and CYVE 1 (low dose cytarabine+high dose cytarabine+etoposide+methotrexate, 12 cases) stages. Multinomial Logistic regression analysis showed that the age over 10 years old ( OR=0.54,95% CI 0.30-0.93), tumor lysis syndrome before chemotherapy ( OR=0.48,95% CI 0.27-0.84) and grade Ⅲ-Ⅳ myelosuppression after chemotherapy ( OR=0.55,95% CI 0.33-0.91)were independent risk factors for chemotherapy delay.The 3-year OS rate and the 3-year EFS rate of children with Burkitt lymphoma in the delayed chemotherapy group were lower than those in the non-delayed chemotherapy group ((79.4±4.9)% vs. (94.2±1.1)%, (80.2±4.8)% vs. (92.0±1.2)%,both P<0.05). The 3-year OS rate of the group C with chemotherapy delay >7 days (42 cases) was lower than that of the group with chemotherapy delay of 1-7 days (22 cases) and the non-delay group (399 cases) ((76.7±6.9)% vs. (81.8±8.2)% vs. (92.7±1.3)%, P=0.002).The 3-year OS rate of the chemotherapy delay group (9 cases) in the COP (vincristine+cyclophosphamide+prednisone) phase was lower than that of the non-chemotherapy delay group (454 cases) ((66.7±15.7)% vs. (91.3±1.4)%, P=0.005). Similarly, the 3-year OS rate of the chemotherapy delay group (11 cases) in the COPADM1 phase was lower than that of the non-chemotherapy delay group (452 cases) ((63.6±14.5)% vs. (91.5±1.3)%, P=0.001). Conclusions:The delayed chemotherapy was related to the age over 10 years old, tumor lysis syndrome before chemotherapy and grade Ⅲ-Ⅳ myelosuppression after chemotherapy in pediatric BL. There is a significant relationship between delayed chemotherapy and prognosis of BL in children.

Objective:To study a method for automatically extracting acupuncture and moxibustion prescriptions from clinical literature to assist data mining of acupuncture and moxibustion prescriptions; To support clinical research and decision-making in acupuncture and moxibustion treatment.Methods:The Chinese journal articles on clinical trials of acupuncture and moxibustion in CNKI from January 1, 1992 to December 31, 2022 were searched. The titles and abstracts of 750 articles were randomly selected and manually labeled. The three main entities of disease name, acupuncture and moxibustion method and acupoint of acupuncture and moxibustion prescription were selected. From the data set, 70% was selected as the training set, 15% as the validation set, and 15% as the test set for the experiment. The extraction of acupuncture prescriptions was considered a sequence labeling task. A model for automatic extraction of acupuncture prescriptions was built using a pretrained language model (PLM), and four different PLMs were selected to compare their entity recognition effects. The impact of negative sampling and label smoothing training techniques on the model was further investigated.Results:The model based on eHealth had the highest F1 scores (92.84). During training, if only negative sampling technology was used, F1 value increased to 93.53; if only label smoothing was used, F1 value increased to 93.64; if negative sampling and label smoothing were used simultaneously, F1 value increased to 94.28, an increase of 1.55%. Conclusions:This study proposes a fast and accurate model for extracting acupuncture and moxibustion prescriptions. The research shows that the model recognition effect based on eHealth in the biomedical field is the best, and the recognition effect of the model can be further improved by using negative sampling and label smoothing training techniques.