Ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry/mass spectrometry(UPLC-Q-TOF-MS/MS), network pharmacology, and animal experiments were integrated o explore the blood glucose-lowering effects and mechanisms of Poria aqueous extract. Firstly, the active components of Poria aqueous extract were identified by UPLC-Q-TOF-MS/MS. Subsequently, network pharmacology was employed to predict the blood glucose-lowering components and mechanisms of Poria aqueous extract. Finally, a rat model of diabetes mellitus, 16S rDNA sequencing, and Western blot were employed to investigate the blood glucose-lowering effect and mechanism of Poria aqueous extract. A total of 39 triterpenoids were identified in the Poria aqueous extract, among them, 25-hydroxypachymic acid, 25α-hydroxytumulosic acid, 16α-hydroxytrametenolic acid, polyporenic acid C, and tumulosic acid may be the main active ingredients for treating diabetes. The Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis revealed that Poria might exert its therapeutic effects through multiple pathways such as NOD-like receptor signaling pathway, nuclear factor-kappa B(NF-κB) signaling pathway, and tumor necrosis factor(TNF) signaling pathway. The results of animal experiments demonstrated that Poria aqueous extract significantly reduced the levels of blood glucose and lipids and regulated the intestinal flora in diabetic rats. The main affected taxa included g＿Escherichia-Shigella, g＿Corynebacterium, g＿Prevotella＿9, g＿Prevotellaceae＿UCG-001, and g＿Bacteroidota＿unclassified. In addition, Poria aqueous extract lowered the levels of D-lactic acid and lipopolysaccharide, alleviated colonic mucosal damage, significantly down-regulated the protein levels of NOD-like receptor pyrin domain-containing protein 3(NLRP3), NF-κB, and TNF-α, and significantly up-regulated the protein levels of zonula occludens 1 and occludin in diabetic rates. Poria aqueous extract may play a role in treating diabetes mellitus by repairing the intestinal flora disturbance, protecting the intestinal barrier function, and inhibiting the NF-κB/NLRP3 signaling pathway. The results provide a scientific basis for clinical application and expansion of indications of Poria.
Animals ; Rats ; Network Pharmacology ; Tandem Mass Spectrometry ; Male ; Drugs, Chinese Herbal/pharmacology* ; Chromatography, High Pressure Liquid ; Blood Glucose/drug effects* ; Rats, Sprague-Dawley ; Hypoglycemic Agents/administration & dosage* ; Poria/chemistry* ; Diabetes Mellitus, Experimental/metabolism* ; NF-kappa B/genetics* ; Gastrointestinal Microbiome/drug effects* ; Humans

OBJECTIVE: To analyze the Epstein-Barr virus (EBV) load in different lymphocyte subsets, as well as clinical characteristics and outcomes in patients with hematologic malignancies experiencing EBV reactivation. METHODS: Peripheral blood samples from patients were collected. B, T, and NK cells were isolated sorting with magnetic beads by flow cytometry. The EBV load in each subset was quantitated by real-time quantitative polymerase chain reaction (RT-qPCR). Clinical data were colleted from electronic medical records. Survival status was followed up through outpatient visits and telephone calls. Statistical analyses were performed using SPSS 25.0. RESULTS: A total of 39 patients with hematologic malignancies were included, among whom 35 patients had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). The median time to EBV reactivation was 4.8 months (range: 1.7-57.1 months) after allo-HSCT. EBV was detected in B, T, and NK cells in 20 patients, in B and T cells in 11 patients, and only in B cells in 4 patients. In the 35 patients, the median EBV load in B cells was 2.19×10⁴ copies/ml, significantly higher than that in T cells (4.00×10³ copies/ml, P <0.01) and NK cells (2.85×10² copies/ml, P <0.01). Rituximab (RTX) was administered for 32 patients, resulting in EBV negativity in 32 patients with a median time of 8 days (range: 2-39 days). Post-treatment analysis of 13 patients showed EBV were all negative in B, T, and NK cells. In the four non-transplant patients, the median time to EBV reactivation was 35 days (range: 1-328 days) after diagnosis of the primary disease. EBV was detected in one or two subsets of B, T, or NK cells, but not simultaneously in all three subsets. These patients received a combination chemotherapy targeting at the primary disease, with 3 patients achieving EBV negativity, and the median time to be negative was 40 days (range: 13-75 days). CONCLUSION In hematologic malignancy patients after allo-HSCT, EBV reactivation commonly involves B, T, and NK cells, with a significantly higher viral load in B cells compared to T and NK cells. Rituximab is effective for EBV clearance. In non-transplant patients, EBV reactivation is restricted to one or two lymphocyte subsets, and clearance is slower, highlighting the need for prompt anti-tumor therapy.
Humans ; Hematologic Neoplasms/virology* ; Herpesvirus 4, Human/physiology* ; Epstein-Barr Virus Infections ; Hematopoietic Stem Cell Transplantation ; Virus Activation ; Lymphocyte Subsets/virology* ; Flow Cytometry ; Killer Cells, Natural/virology* ; Male ; Female ; B-Lymphocytes/virology* ; Viral Load ; Adult ; T-Lymphocytes/virology* ; Middle Aged

OBJECTIVE: To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. METHODS: In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. RESULTS: Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. CONCLUSION AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.
Podocytes/metabolism* ; Animals ; Diabetic Nephropathies/metabolism* ; Saponins/therapeutic use* ; Triterpenes/therapeutic use* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Male ; Rats, Sprague-Dawley ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Endoribonucleases/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; Rats ; Diabetes Mellitus, Experimental/complications* ; Endoplasmic Reticulum/metabolism* ; Multienzyme Complexes

OBJECTIVE: To explore the mechanism of colon dialysis with Yishen Decoction (YS) in improving the autophagy disorder of intestinal epithelial cells in chronic renal failure (CRF) in vivo and in vitro. METHODS: Thirty male SD rats were randomly divided into normal, CRF, and colonic dialysis with YS groups by a random number table method (n=10). The CRF model was established by orally gavage of adenine 200 mg/(kg•d) for 4 weeks. CRF rats in the YS group were treated with colonic dialysis using YS 20 g/(kg•d) for 14 consecutive days. The serum creatinine (SCr) and urea nitrogen (BUN) levels were detected by enzyme-linked immunosorbent assay. Pathological changes of kidney and colon tissues were observed by hematoxylin and eosin staining. Autophagosome changes in colonic epithelial cells was observed with electron microscopy. In vitro experiments, human colon cancer epithelial cells (T84) were cultured and divided into normal, urea model (74U), YS colon dialysis, autophagy activator rapamycin (Ra), autophagy inhibitor 3-methyladenine (3-MA), and SIRT1 activator resveratrol (Re) groups. RT-PCR and Western blot were used to detect the mRNA and protein expressions of zonula occludens-1 (ZO-1), Claudin-1, silent information regulator sirtuin 1 (SIRT1), LC3, and Beclin-1 both in vitro and in vivo. RESULTS: Colonic dialysis with YS decreased SCr and BUN levels in CRF rats (P<0.05), and alleviated the pathological changes of renal and colon tissues. Expressions of SIRT1, ZO-1, Claudin-1, Beclin-1, and LC3II/I were increased in the YS group compared with the CRF group in vivo (P<0.05). In in vitro study, compared with normal group, the expressions of SIRT1, ZO-1, and Claudin-1 were decreased, and expressions of Beclin-1, and LC3II/I were increased in the 74U group (P<0.05). Compared with the 74U group, expressions of SIRT1, ZO-1, and Claudin-1 were increased, whereas Beclin-1, and LC3II/I were decreased in the YS group (P<0.05). The treatment of 3-MA and rapamycin regulated autophagy and the expression of SIRT1. SIRT1 activator intervention up-regulated autophagy as well as the expressions of ZO-1 and Claudin-1 compared with the 74U group (P<0.05). CONCLUSION Colonic dialysis with YS could improve autophagy disorder and repair CRF intestinal mucosal barrier injury by regulating SIRT1 expression in intestinal epithelial cells.
Animals ; Sirtuin 1/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Autophagy/drug effects* ; Male ; Intestinal Mucosa/drug effects* ; Rats, Sprague-Dawley ; Epithelial Cells/metabolism* ; Colon/drug effects* ; Humans ; Kidney Failure, Chronic/drug therapy* ; Signal Transduction/drug effects* ; Renal Dialysis ; Rats ; Kidney/drug effects*

Aim To investigate the therapeutic effect of newly formulated Tadalafil tablets on liver fibrosis in mice induced by carbon tetrachloride(CCl4)and its impact on the activation of hepatic stellate cells(HSCs).Methods Liver fibrosis model was estab-lished by intraperitoneally injecting 20％CCl4 corn oil solution twice a week for eight weeks.After four weeks of modeling,the treatment group was administered ei-ther the newly formulated Tadalafil tablets(1.0 mg·kg-1)or the Cialis(2.5 mg·kg-1)via gavage for the remaining four weeks.We assessed the effects of Tadalafil on collagen deposition,tissue structural dam-age,and HSCs activation markers in the fibrotic liver of mice using serum biochemical analysis,histopathologi-cal staining,and Western blotting following the treat-ment period.LX-2 cells were cultured and treated with tadalafil after TGF β1 stimulation,and the effects of tadalafil on LX-2 cell activation were assessed via Western blot.Results Compared to the normal mice,the model group mice exhibited a significantly higher liver-specific index,increased liver function indicators,and notable hepatocyte necrosis.Additionally,liver lobules were damaged,accompanied by severe infiltra-tion of inflammatory cells.Both smooth muscle actin(α-SMA)and fibronectin(Fn)were elevated,serving as markers of HSCs activation.As a result of treatment with the newly formulated Tadalafil tablets,liver tissue damage was significantly reduced,transaminase levels decreased,necrosis and inflammatory cell infiltration were reduced,and collagen fiber deposition was allevia-ted,and α-SMA and Fn expression was reduced.It was worth noting that low-dose newly formulated Tadalafil tablets were found to be as effective as high-dose Cia-lis.In a cellular model,Tadalafil significantly inhibited the activation of LX-2 cells and reduced the expression of proteins related to cell activation.Conclusions The newly formulated Tadalafil tablets can significantly inhibit HSCs activation,reduce extracellular matrix(ECM)deposition,improve liver fibrosis and liver function damage caused by CCl4.This new formulation offers a significant advantage over Cialis in terms of ef-fectiveness,with a lower effective dose.

Aim To investigate the effects of SAHA on the expression of P-gp and the pharmacokinetic pa-rameters of its substrate phenytoin sodium in rats under hypoxic environments.Methods Wistar rats were randomly divided into the normioxic group,the hypoxic model group,and the low-,medium-and high-dose vorinostat(SAHA)groups.Liver tissues were col-lected,and the expression levels of P-gp and HDAC5 were detected by Real-time PCR and Western blot.The morphological changes of liver tissues were ob-served by HE staining.Following intragastric adminis-tration of 50 mg·kg-1 phenytoin sodium to each group,blood samples were collected,and the plasma concentration of phenytoin sodium was determined u-sing UFLC-MS/MS to calculate pharmacokinetic pa-rameters.Results Compared with the normoxic group,the expression of HDAC5 in the liver tissues of hypoxia model rats increased,while the expression of P-gp decreased.After SAHA treatment,HDAC5 expression decreased,and P-gp expression increased.Among the SAHA groups,the medium-dose group showed the most significant effect,and HE staining re-sults indicated that this concentration did not cause damage to rat liver tissues.Compared with the normox-ic group,the AUC,Cmax,and T1/2 of phenytoin sodium in hypoxia model rats were significantly raised.After administration of the medium dose of SAHA,the AUC,Cmax,MRT,and T1/2 were significantly reduced,while CLZ/r was significantly increased.Conclusions Un-der hypoxic environments,the expression of P-gp in rat liver tissue is significantly downregulated,leading to increased systemic exposure of phenytoin,reduced clearance,and consequently elevated blood concentra-tions,raising the risk of central nervous system toxici-ty.In contrast,SAHA suppresses HDAC5 expression,thereby activating P-gp transcription and enhancing its efflux function.This results in decreased systemic ex-posure and improved clearance of phenytoin,signifi-cantly reducing drug accumulation in body and ulti-mately lowering the risk of adverse effects.

AIM To establish the quantitative models for gallic acid,mononucleoside,loganin,resveratrol,and rhein in Yishen Xiezhuo Mixture.METHODS HPLC was adopted in the content determination of various effective components,after which the near-infrared spectroscopy(NIRS)data were collected in 128 batches of samples and pretreatment was conducted,competitive adaptive reweighting sampling(CARS)algorithm was used for screening wavelength,partial least square method(PLS)regression analysis was performed.RESULTS There were no significant differences between the predicted values obtained by PLS models and measured values obtained by HPLC for various effective components(P＞0.05).CONCLUSION The quantitative models established by NIRS combined with chemometrics display good predictive performance,which can be used for the rapid determination of effective components in Yishen Xiezhuo Mixture,and provide a reference for the rapid monitoring of other traditional Chinese medicine preparations in production processes.

AIM To establish the preparation process and quality standard for Zhenggu Pills.METHODS With decoction time,decoction frequency and water addition as influencing factors,comprehensive score for extract yield and transfer rates of epicatechin and naringin as an evaluation index,the decoction process was optimized by orthogonal test.With sugarless paste relative density,medicinal powder fineness,sugarless paste-corn starch ratio,drying temperature and drying time as influencing factors,soft material traits,pill formability,moisture and disintegration time limit as evaluation indices,the formability process was optimized by single factor test.TLC was adopted in the qualitative identification of Dipsaci Radix,salt-processed Psoraleae Fructus,cooked Rhei Radix et Rhizoma and Notoginseng Radix et Rhizoma.HPLC was used for the content determination of paeoniflorin and naringin.RESULTS The optimal decoction process was determined to be 0.5 h for decoction time,two times for decoction frequency,and 10 times for water addition,the comprehensive score was 0.93.The optimal formability process was determined to be 1.21-1.22 for sugarless paste relative density,80 mesh for medicinal powder fineness,1∶0.17-1∶0.18 for sugarless paste-corn starch ratio,70 ℃ for drying temperature,and 24 h for drying time,good soft material traits and pill formability were observable,and moisture and disintegration time limit accored with 2020 edition of Chinese Pharmacopoeia requirements.The TLC spots were clear without negative interference.Two constituents showed good linear relationships within 61.30-490.41 μg/mL(r=0.999 8)and 3.27-26.18 μg/mL(r=0.999 8),whose average recoveries were 100.15％and 98.15％with the RSDs of 0.55％and 2.30％,respectively.CONCLUSION This stable,reliable and specific method can be used for the production and quality evaluation of Zhenggu Pills.

Objective:To construct a key item checklist for the Mini-HTA report specification,providing scientific guidance for drafting each section of Mini-HTA research reports,enhancing their standardization,scientific rigor,and completeness,thereby improving the efficiency and quality of health decision-making.Methods:Based on preliminary literature review and qualitative systematic review,a pool of problem items for the Mini-HTA report specification was formed.Delphi questionnaires were distributed,and the Delphi technique was employed through two rounds of expert consultation to optimize and select key items.Results:Through two rounds of Delphi expert consultation,the initial Mini-HTA report specification item checklist was screened,integrated,and supplemented.A finalized key item checklist was constructed,comprising 8 first-level items(Title,Abstract,Introduction,Methods,Results,Discussion,Conclusion,and Other Relevant Information)and 48 second-level items.Conclusion:The constructed key item checklist for the Mini-HTA report specification provides scientific guidance for drafting Mini-HTA research reports.It helps enhance the standardization and transparency of the assessment process and the reliability of results,thereby optimizing the efficiency and quality of health decision-making.

AIM To establish the preparation process and quality standard for Zhenggu Pills.METHODS With decoction time,decoction frequency and water addition as influencing factors,comprehensive score for extract yield and transfer rates of epicatechin and naringin as an evaluation index,the decoction process was optimized by orthogonal test.With sugarless paste relative density,medicinal powder fineness,sugarless paste-corn starch ratio,drying temperature and drying time as influencing factors,soft material traits,pill formability,moisture and disintegration time limit as evaluation indices,the formability process was optimized by single factor test.TLC was adopted in the qualitative identification of Dipsaci Radix,salt-processed Psoraleae Fructus,cooked Rhei Radix et Rhizoma and Notoginseng Radix et Rhizoma.HPLC was used for the content determination of paeoniflorin and naringin.RESULTS The optimal decoction process was determined to be 0.5 h for decoction time,two times for decoction frequency,and 10 times for water addition,the comprehensive score was 0.93.The optimal formability process was determined to be 1.21-1.22 for sugarless paste relative density,80 mesh for medicinal powder fineness,1∶0.17-1∶0.18 for sugarless paste-corn starch ratio,70 ℃ for drying temperature,and 24 h for drying time,good soft material traits and pill formability were observable,and moisture and disintegration time limit accored with 2020 edition of Chinese Pharmacopoeia requirements.The TLC spots were clear without negative interference.Two constituents showed good linear relationships within 61.30-490.41 μg/mL(r=0.999 8)and 3.27-26.18 μg/mL(r=0.999 8),whose average recoveries were 100.15％and 98.15％with the RSDs of 0.55％and 2.30％,respectively.CONCLUSION This stable,reliable and specific method can be used for the production and quality evaluation of Zhenggu Pills.