Discovery of Natural Products Alleviating Renal Fibrosis with a Viscosity-Responsive Molecular Probe.

Renal fibrosis poses a significant threat to individuals suffering from chronic progressive kidney disease. Given the absence of effective medications for treating renal fibrosis, it becomes crucial to assess the extent of fibrosis in real time and explore the development of novel drugs with substantial therapeutic benefits. Due to the accumulation of renal tissue damage and the uncontrolled deposition of fibrotic matrix during the course of the disease, there is an increase in viscosity both intracellularly and extracellularly. Therefore, a viscosity-sensitive near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe, BDP-KY, was developed to detect aberrant changes in viscosity during fibrosis. Furthermore, BDP-KY has been applied to screen the effective components of herbal medicine, rhubarb, resulting in the identification of potential antirenal fibrotic compounds such as emodin-8-glucoside and chrysophanol 8-O-glucoside. Ultrasound, PA, and NIRF imaging of a unilateral uretera obstruction mice model show that different concentrations of emodin-8-glucoside and chrysophanol 8-O-glucoside effectively reduce viscosity levels during the renal fibrosis process. The histological results showed a significant decrease in fibrosis factors α-smooth muscle actin and collagen deposition. Combining these findings with their pharmacokinetic characteristics, these compounds have the potential to fill the current market gap for effective antirenal fibrosis drugs. This study demonstrates the potential of BDP-KY in the evaluation of renal fibrosis, and the two identified active components from rhubarb hold great promise for the treatment of renal fibrosis.

Blockade of spinal dopamine D1/D2 receptor heteromers by levo-Corydalmine suppressed calcium signaling cascade in spinal neurons to alleviate bone cancer pain in rats.

Background: Dopamine receptors have been reported to be involved in pain, while the exact effects and mechanism in bone cancer pain have not been fully explored. Methods: Bone cancer pain model was created by implanting walker 256 mammary gland carcinoma into right tibia bone cavity. Primary cultured spinal neurons were used for in vitro evaluation. FLIPR, western-blot, immunofluorescence, and Co-IP were used to detect cell signaling pathway. Results: Our results indicated that spinal dopamine D1 receptor (D1DR) and spinal dopamine D2 receptor (D2DR) could form heteromers in TCI rats, and antagonizing spinal D1DR and D2DR reduced heteromers formation and alleviated TCI-induced bone cancer pain. Further results indicated that D1DR or D2DR antagonist induced antinociception in TCI rats could be reversed by D1DR, D2DR, and D1/D2DR heteromer agonists. And Gq, IP3, and PLC inhibitors also attenuated TCI-induced bone cancer pain. In vitro results indicated that D1DR or D2DR antagonist decreased the Ca2+ oscillations upregulated by D1DR, D2DR, and D1/D2DR heteromer agonists in activated primary cultured spinal neurons. Moreover, inhibition of D1/D2DR heteromers induced antinociception in TCI rats was partially mediated by the CaMKII and MAPKs pathway. In addition, a natural compound levo-Corydalmine (l-CDL), could inhibit D1/D2DR heteromers and attenuate bone cancer pain. Results: Inhibition of spinal D1/D2DR heteromers via l-CDL decreases excitability in spinal neurons, which might present new therapeutic strategy for bone cancer pain.

A novel method for evaluating pseudoallergy based on ß-hexosaminidase and its application for traditional Chinese medicine injections.

Pseudoallergy is a typical and common adverse drug reaction to injections, especially in traditional Chinese medicine injections (TCMIs). At present, the evaluation methods for pseudoallergy include cell methods in vitro and animal methods in vivo. The mast cell evaluation method based on the ß-hexosaminidase (ß-Hex)-catalyzed substrate, 4-nitrophenyl-ß-N-acetyl-D-glucosaminide (4-NPG), is an important method for the evaluation of drug-induced pseudoallergy, but it is prone to false positive results and has insufficient sensitivity. In this study, a novel ß-Hex evaluation system with rat basophilic leukemia-2H3 cells based on high-performance liquid chromatography-fluorescence detection (HPLC-FLD) was established, which effectively increased the sensitivity and avoided false positive results. Cell viabilities were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. In addition, a method for the determination of histamine, which is another indicator in the development of pseudoallergy, was established to validate the above method. The results of this novel method indicated that two TCMIs (Shuxuening injection and Shenqi Fuzheng injection), which were considered to be pseudoallergenic using 4-NPG, were not pseudoallergenic. Overall, the novel ß-Hex/HPLC-FLD evaluation system using Rat basophilic leukemia-2H3 cells established was effective and precise. It could be used for the evaluation of pseudoallergic reactions caused by TCMIs and other injections.

Discovering a New Drug Against Acute Kidney Injury by Using a Tailored Photoacoustic Imaging Probe.

Acute kidney injury (AKI) has become an increasing concern for patients due to the widespread clinical use of nephrotoxic drugs. Currently, the early diagnosis of AKI is still challenging and the available therapeutic drugs cannot meet the clinical demand. Herein, this work has investigated the key redox couple involved in AKI and develops a tailored photoacoustic (PA) imaging probe (AB-DiOH) which can reversibly respond to hypochlorite (ClO-)/glutathione (GSH) with high specificity and sensitivity. This probe enables the real-time monitoring of AKI by noninvasive PA imaging, with better detection sensitivity than the blood test. Furthermore, this probe is utilized for screening nephroprotective drugs among natural products. For the first time, astragalin is discovered to be a potential new drug for the treatment of AKI. After oral administration, astragalin can be efficiently absorbed by the animal body, alleviate kidney injury, and meanwhile induce no damage to other normal tissues. The treatment mechanism of astragalin has also been revealed to be the simultaneous inhibition of oxidative stress, ferroptosis, and cuproposis. The developed PA imaging probe and the discovered drug candidate provide a promising new tool and strategy for the early diagnosis and effective treatment of AKI.

Illumination of Hydroxyl Radical in Kidney Injury and High-Throughput Screening of Natural Protectants Using a Fluorescent/Photoacoustic Probe.

The hydroxyl radical (•OH) is shown to play a crucial role in the occurrence and progression of acute kidney injury (AKI). Therefore, the development of a robust •OH probe holds great promise for the early diagnosis of AKI, high-throughput screening (HTS) of natural protectants, and elucidating the molecular mechanism of intervention in AKI. Herein, the design and synthesis of an activatable fluorescent/photoacoustic (PA) probe (CDIA) for sensitive and selective imaging of •OH in AKI is reported. CDIA has near-infrared fluorescence/PA channels and fast activation kinetics, enabling the detection of the onset of •OH in an AKI model. The positive detection time of 12 h using this probe is superior to the 48-hour detection time for typical clinical assays, such as blood urea nitrogen and serum creatinine detection. Furthermore, a method is established using CDIA for HTS of natural •OH inhibitors from herbal medicines. Puerarin is screened out by activating the Sirt1/Nrf2/Keap1 signaling pathway to protect renal cells in AKI. Overall, this work provides a versatile and dual-mode tool for illuminating the •OH-related pathological process in AKI and screening additional compounds to prevent and treat AKI.

Ruscogenin Alleviates Myocardial Ischemia via Myosin IIA-Dependent Mitochondrial Fusion and Fission Balance.

Ruscogenin (RUS), a major effective steroidal sapogenin derived from Ophiopogon japonicas, has been reported to alleviate myocardial ischemia (MI), but its cardioprotective mechanism is still not completely clear. In this study, we observed that RUS markedly reduced MI-induced myocardial injury, as evidenced by notable reductions in infarct size, improvement in biochemical markers, alleviation of cardiac pathology, amelioration of mitochondrial damage, and inhibition of myocardial apoptosis. Moreover, RUS notably suppressed oxygen-glucose deprivation (OGD)-triggered cell injury and apoptosis. Notably, RUS demonstrated a considerable decrease of the interaction between myosin IIA and F-actin, along with the restoration of mitochondrial fusion and fission balance. We further confirmed that the effects of RUS on MI were mediated by myosin IIA using siRNA and overexpression techniques. The inhibition of myosin IIA resulted in a significant improvement of mitochondrial fusion and fission imbalance, while simultaneously counteracting the beneficial effects of RUS. By contrast, overexpression of myosin IIA aggravated the imbalance between mitochondrial fusion and fission and partially weakened the protection of RUS. These findings suggest that myosin IIA is essential or even a key functional protein in the cardioprotection of RUS. Overall, our results have elucidated an undiscovered mechanism involving myosin IIA-dependent mitochondrial fusion and fission balance for treating MI. Furthermore, our study has uncovered a novel mechanism underlying the protective effects of RUS.

Development and validation of a prediction model for mechanical ventilation based on comorbidities in hospitalized patients with COVID-19.

Background: Timely recognition of respiratory failure and the need for mechanical ventilation is crucial in managing patients with coronavirus disease 2019 (COVID-19) and reducing hospital mortality rate. A risk stratification tool could assist to avoid clinical deterioration of patients with COVID-19 and optimize allocation of scarce resources. Therefore, we aimed to develop a prediction model for early identification of patients with COVID-19 who may require mechanical ventilation. Methods: We included patients with COVID-19 hospitalized in United States. Demographic and clinical data were extracted from the records of the Healthcare Cost and Utilization Project State Inpatient Database in 2020. Model construction involved the use of the least absolute shrinkage and selection operator and multivariable logistic regression. The model's performance was evaluated based on discrimination, calibration, and clinical utility. Results: The training set comprised 73,957 patients (5,971 requiring mechanical ventilation), whereas the validation set included 10,428 (887 requiring mechanical ventilation). The prediction model incorporating age, sex, and 11 other comorbidities (deficiency anemias, congestive heart failure, coagulopathy, dementia, diabetes with chronic complications, complicated hypertension, neurological disorders unaffecting movement, obesity, pulmonary circulation disease, severe renal failure, and weight loss) demonstrated moderate discrimination (area under the curve, 0.715; 95% confidence interval, 0.709-0.722), good calibration (Brier score = 0.070, slope = 1, intercept = 0) and a clinical net benefit with a threshold probability ranged from 2 to 34% in the training set. Similar model's performances were observed in the validation set. Conclusion: A robust prognostic model utilizing readily available predictors at hospital admission was developed for the early identification of patients with COVID-19 who may require mechanical ventilation. Application of this model could support clinical decision-making to optimize patient management and resource allocation.

Generation of a high-affinity DNA aptamer for on-site screening of toxic aristolochic acid I in herbal medicines and botanical products.

Aristolochic Acid I (AAI) is an environmental and foodborne toxin found in the Aristolochia and Asarum species of plants that are widespread all over the world. Therefore, there is an urgent need to develop a sensitive and specific biosensor for identifying AAI. Aptamers as a powerful biorecognition element provide the most viable options for solving this problem. In this study, we used library-immobilized SELEX to isolate an AAI-specific aptamer with a KD value of 86 ± 13 nM. To verify the practicability of the selected aptamer, a label-free colorimetric aptasensor was designed. This aptasensor exhibited a low detection limit of 225 nM. Besides, it had been further applied for the determination of AAI in real samples and the recoveries ranged from 97.9% to 102.4%. In the future, AAI aptamer will provide a promising tool for safety evaluation in various fields of agriculture, food, and medication.

Analysis of 12 Chemical Compounds And Pattern Recognition of Different Parts of Angelicae Sinensis Radix by Liquid Chromatography-Tandem Mass Spectrometry And Chemometrics Methods.

To evaluate the quality and quantify bioactive constituents in different parts of Angelicae Sinensis Radix, an efficient, high-speed, high-sensitivity high-performance liquid chromatography and triple quadrupole mass spectrometry method was used for simultaneous detection of 12 chemical compounds including L-tryptophan, chlorogenic acid, caffeic acid, ferulic acid, isoferulic acid, senkyunolide I, guanosine, proline, L-glutamine, γ-aminobutyric acid, glutamic acid, and arginine in 52 batches of Angelicae Sinensis Radix from Gansu, China. The established methods were validated by good linearity (R2≥0.9921), limits of detection (0.0001-0.0156 µg/mL), limits of quantitation (0.0006-0.0781 µg/mL), stability (RSD≤7.77%), repeatability (RSD≤6.79%), intra- and interday precisions (RSD≤6.00% and RSD≤6.39%, respectively) and recovery (90.90-107.16%). According to the quantitative results, the contents of the hydrophilic compounds were higher in the head, while the medium and weak polar components were mainly concentrated in the tail. Finally, principal component analysis results revealed that Angelicae Sinensis Radix could be divided into different medicinal sites based on polar components such as amino acids, nucleosides. The combination of liquid chromatography-tandem mass spectrometry and principal component analysis is a simple and reliable method for pattern recognition and quality evaluation of Angelicae Sinensis Radix.

Impact of Social Deprivation on Hospitalization and Intensive Care Unit Admission among COVID-19 Patients: A Systematic Review and Meta-Analysis.

Background: The coronavirus disease 2019 (COVID-19) pandemic has disproportionately affected socially disadvantaged groups; however, the association between socioeconomic status and healthcare utilization among COVID-19 patients remains unclear. Therefore, a systematic review and meta-analysis was conducted to assess the association between socioeconomic status and hospitalization and intensive care unit admission among COVID-19 patients. Methods: PubMed, Embase, and the Cochrane Register of Controlled Trials were searched for relevant literature (updated to Jun 2022). Studies that investigated the association of social deprivation with hospitalization and intensive care unit admission in COVID-19 patients were included. The primary outcomes included risk of hospitalization and intensive care unit admission, measured by odds ratio. Results: Eleven studies covering 2,423,095 patients were included in the meta-analysis. Socially disadvantaged patients had higher odds of hospitalization in comparison to socially advantaged patients (odds ratio 1.25, 95% confidence interval: 1.14 to 1.38; P<0.01). The odds of intensive care unit admission among more deprived patients was not significantly different from that of less deprived patients (odds ratio 1.03, 95% confidence interval: 0.78 to 1.35; P=0.85). These findings were proven robust through subgroup and sensitivity analyses. Conclusion: Socially disadvantaged populations have higher odds of hospitalization if they become infected with COVID-19. More effective medical support and interventions for these vulnerable populations are required to reduce inequity in healthcare utilization and alleviate the burden on healthcare systems.

Parallel use of pluripotent human stem cell lung and heart models provide new insights for treatment of SARS-CoV-2.

SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems. One-sentence summary: Rational treatment strategies for SARS-CoV-2 derived from human PSC models.

Parallel use of pluripotent human stem cell lung and heart models provide new insights for treatment of SARS-CoV-2

SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems. One-sentence summaryRational treatment strategies for SARS-CoV-2 derived from human PSC models

Small-Molecule Photoacoustic Imaging Probe with Aggregation-Enhanced Amplitude for Real-Time Visualization of Acute Kidney Injury.

Acute kidney injury (AKI) has become a growing issue for patients with the extensive use of all kinds of drugs in clinic. Photoacoustic (PA) imaging provides a noninvasive and real-time imaging method for studying kidney injury, but it has inherent shortages in terms of high background signal and low detection sensitivity for exogenous imaging agents. Intriguingly, J-aggregation offers to tune the optical properties of the dyes, thus providing a platform for developing new PA probes with desired performance. In this study, a small-molecule PA probe (BDP-3) was designed and synthesized. We serendipitously discovered that BDP-3 can transform into renal clearable nanoaggregates under physiological conditions. The hydrodynamic diameter of the BDP-3 increased from 0.64 ± 0.11 to 3.74 ± 0.39 nm when the content of H2O increased from 40 to 90%. In addition, it was surprising that such a transforming process can significantly enhance its PA amplitude (2.06-fold). On this basis, PA imaging with BDP-3 was applied as a new method for the noninvasive detection of AKI induced by anticancer drugs, traditional Chinese medicine, and clinical contrast agents in animal models and exhibited higher sensitivity than the conventional serum index test, demonstrating great potential for further clinical diagnostic applications.

Anti-inflammatory effects of Abelmoschus manihot (L.) Medik. on LPS-induced cystitis in mice: potential candidate for cystitis treatment based on classic use.

Abelmoschus manihot (L.) Medik. (A. manihot) is a traditional Chinese herbal medicine with a variety of pharmacological properties. It was first recorded in Jiayou Materia Medica dating back to the Song dynasty to eliminate urinary tract irritation by clearing away heat and diuretic effect. However, its pharmacological action on urinary tract infections has not been investigated. The present study aims to evaluate the anti-inflammatory activity of A. manihot on a mouse model of lipopolysaccharide (LPS)-induced cystitis. The results showed that A. manihot decreased white blood cell (WBC) count in urine sediments of the cystitis mice, alleviated bladder congestion, edema, as well as histopathological damage, reduced the expression levels of tumor necrosis factor-α, interleukin-6, and interleukin-1ß simultaneously. Moreover, A. manihot administration significantly downregulated the expression levels of TLR4, MYD88, IκBα, p-IκBα, NF-κB p65, and p-NF-κB p65 in LPS-induced cystitis mice. These findings demonstrated the protective effect of A. manihot against LPS-induced cystitis, which is attributed to its anti-inflammatory profile by suppressing TLR4/MYD88/NF-κB pathways. Our results suggest that A. manihot could be a potential candidate for cystitis treatment.

Renal-clearable and biodegradable black phosphorus quantum dots for photoacoustic imaging of kidney dysfunction.

The kidney is a vital organ and susceptible to various diseases. Photoacoustic (PA) imaging provides a powerful technique for studying kidney dysfunction, for which many smart photoacoustic imaging agents have been developed. But the complete clearance of the introduced contrast agents after imaging remains to be challenging, leading to long-term toxicity concerns. In this study, we synthesized black phosphorous quantum dots (BPQDs) with ultra-small size (1.74 ± 0.23 nm after surface modification) and strong PA signal for imaging kidney dysfunction. Importantly, the renal-clearance property and biodegradability of the developed BPQDs help circumvent the long-term toxicity issue for in vivo studies. Based on these BPQDs, both acute kidney injury and chronic kidney disease were successfully detected in the living mice by PA imaging, with higher detection sensitivity than the clinical serum indices examination method. This BPQDs-based PA imaging method should have a promising potential for the early diagnosis of kidney dysfunction in clinic.

Isoorientin protects lipopolysaccharide-induced acute lung injury in mice via modulating Keap1/Nrf2-HO-1 and NLRP3 inflammasome pathways.

Acute lung injury (ALI) is a pulmonary disease with high mortality. The present study investigated the protective effect of isoorientin (ISO) on lipopolysaccharide (LPS)-induced ALI compared with Thalictrum minus L. (TML). The experimental ALI was achieved by LPS via endotracheal drip, ISO and TML (40 mg/kg) were administered orally 1 h prior to LPS. ISO treatment significantly protected mice from ALI and exhibited similar efficacy as TML. Administration of ISO markedly corrected weight loss and improved lung pathological damage caused by LPS. Meanwhile, a decline of lung wet to dry weight (W/D) ratios and total protein in bronchoalveolar fluid (BALF) demonstrated that ISO mitigated pulmonary edema and vascular leakage of ALI mice. Moreover, ISO also signally decreased oxidative stress and suppressed the content of interleukin-6 (IL-6) in BALF. Additionally, ISO significantly promoted the expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and down-regulated kelch-like ECH-associated protein 1 (Keap1). Simultaneously, it suppressed the over-expression of NOD-, LRR- and pyrin domain-containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC) and pro-inflammatory cytokines interleukin IL-1ß (pro-IL-1ß), and inhibited the expression of apoptotic related proteins induced by LPS challenge. Meanwhile, the results of molecular docking indicated the potential ability of ISO as a ligand binding with proteins Keap1, NLRP3 and cleaved-caspase-3 as well. These findings demonstrated that ISO might be one of the bioactive components of TML in the treatment of ALI and provided a rationale for future clinical applications and potential protective strategies for ALI.

Aminoacylase-1 plays a key role in myocardial fibrosis and the therapeutic effects of 20(S)-ginsenoside Rg3 in mouse heart failure.

We previously found that the levels of metabolite N-acetylglutamine were significantly increased in urine samples of patients with heart failure (HF) and in coronary artery ligation (CAL)-induced HF mice, whereas the expression of its specific metabolic-degrading enzyme aminoacylase-1 (ACY1) was markedly decreased. In the current study, we investigated the role of ACY1 in the pathogenesis of HF and the therapeutic effects of 20(S)-ginsenoside Rg3 in HF experimental models in vivo and in vitro. HF was induced in mice by CAL. The mice were administered Rg3 (7.5, 15, 30 mg · kg-1· d-1, i.g.), or positive drug metoprolol (Met, 5.14 mg · kg-1· d-1, i.g.), or ACY1 inhibitor mono-tert-butyl malonate (MTBM, 5 mg · kg-1 · d-1, i.p.) for 14 days. We showed that administration of MTBM significantly exacerbated CAL-induced myocardial injury, aggravated cardiac dysfunction, and pathological damages, and promoted myocardial fibrosis in CAL mice. In Ang II-induced mouse cardiac fibroblasts (MCFs) model, overexpression of ACY1 suppressed the expression of COL3A1 and COL1A via inhibiting TGF-ß1/Smad3 pathway, whereas ACY1-siRNA promoted the cardiac fibrosis responses. We showed that a high dose of Rg3 (30 mg · kg-1· d-1) significantly decreased the content of N-acetylglutamine, increased the expression of ACY1, and inhibited TGF-ß1/Smad3 pathway in CAL mice; Rg3 (25 µM) exerted similar effects in Ang II-treated MCFs. Meanwhile, Rg3 treatment ameliorated cardiac function and pathological features, and it also attenuated myocardial fibrosis in vivo and in vitro. In Ang II-treated MCFs, the effects of Rg3 on collagen deposition and TGF-ß1/Smad3 pathway were slightly enhanced by overexpression of ACY1, whereas ACY1 siRNA partially weakened the beneficial effects of Rg3, suggesting that Rg3 might suppress myocardial fibrosis through ACY1. Our study demonstrates that N-acetylglutamine may be a potential biomarker of HF and its specific metabolic-degrading enzyme ACY1 could be a potential therapeutic target for the prevention and treatment of myocardial fibrosis during the development of HF. Rg3 attenuates myocardial fibrosis to ameliorate HF through increasing ACY1 expression and inhibiting TGF-ß1/Smad3 pathway, which provides some references for further development of anti-fibrotic drugs for HF.

[Analysis on ingredients and metabolites of Gegen Decoction absorbed into blood based on UHPLC-Q-TOF-MS].

This study analyzed the plasma components of Gegen Decoction(GGD) by ultra-high-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UHPLC-Q-TOF-MS), which is expected to serve as a reference for exploring the pharmacodynamic substances of GGD. Female Wistar rats were given(ig) GGD and then plasma samples were collected and analyzed by UHPLC-Q-TOF-MS. The results showed that 42 chemical components were identified: 25 prototypes(14 from Puerariae Lobatae Radix, 6 from Glycyrrhizae Radix et Rhizoma, 3 from Paeoniae Radix Alba, and 2 from Ephedrae Herba) and 17 metabolites(from isoflavonoids in Puerariae Lobatae Radix and Glycyrrhizae Radix et Rhizoma). UHPLC-Q-TOF-MS was employed to achieve rapid analysis of plasma components of GGD, laying a basis for elucidating the therapeutic material basis and mechanism of GGD.

[Comparative study of two high-efficiency methods for purifying spermatogonial stem cells].

Objective: To compare the advantages and disadvantages of the differential attachment method and immunomagnetic bead method for purification of mouse spermatogonial stem cells (mSSCs). Methods: Ten male C57BL/6 mice aged 12～15 days were selected and sacrificed by cervical dislocation. Testes were collected and the seminiferous tubule single cell suspension was obtained by enzymatic digestion. mSSCs were purified by using the differential attachment method and immunomagnetic bead method respectively. Then a detailed comparison of the two methods in terms of cell number, separation efficiency, and impact on cell proliferation and growth was conducted. Results: Both of the methods could isolate and purify stem cells from single cell suspension of mouse seminiferous tubules. mSSCs showed typical grape cluster-like clones in vitro culture, which could be continuously cultured and proliferated for over 3 months in vitro. The testes of 10 mice could obtain 3×105±0.4×105 mSSCs (n=5) by differential attachment method, cell recovery rate (the number of cells after purification/the number of cells of the single cell suspension of seminiferous tubules) was 1.5%±0.1%; 6×105±0.4×105 mSSCs (n= 5) could be obtained by immunomagnetic bead method. The recovery rate was about 3%±0.1%, and the number of stem cells obtained by the immunomagnetic bead method was higher. The stem cells obtained by the differential attachment method were more pure, because the stem cell colonies were preferentially obtained after 5 days of in vitro culture, while the stem cells obtained by the immunomagnetic bead method needed to be cultured for about 10 days before the obvious cell colonies could be observed, but the two types of purification method had no obvious effect on the long-term growth of cells in vitro. Conclusion: Both methods can get high quality mSSCs, but both methods have their own advantages and disadvantages. The differential attachment method is more economical and practical than the other, it does not require special equipment, but the stem cell number obtained is relatively lower and the time needed is longer.

[Role of Puerariae Lobatae Radix in Gegen Decoction for treatment of primary dysmenorrhea].

This study aimed to explore the characteristic role of Puerariae Lobatae Radix(PLR) in Gegen Decoction for the treatment of primary dysmenorrhea(PD). Estrogen(E_2) was combined with oxytocin to establish a mouse model of PD. The mice were randomly divided into a normal group, a model group, a Gegen Decoction group, a PLR-free Gegen Decoction group, a PLR group, and a positive drug group(ibuprofen). Writhing response times and writhing incubation of mice in each group were tested by behavio-ral assessment, and the serum levels of prostaglandin F_(2α)(PGF_(2α)), prostaglandin E_2(PGE_2), E_2, and progesterone(PROG) were detected by ELISA kits. Western blot method was adopted to detect cyclooxygenase-2(COX-2) and estrogen receptor alpha(ER_α) expression levels in uterine tissues. Doppler ultrasound was employed to detect changes in uterine artery blood flow in mice, including peak systolic blood flow velocity(maximum velocity), end-diastolic velocity(minimum velocity), peak systolic blood flow velocity/end-diastolic velocity(S/D), pulsatility index(PI), and resistive index(RI). Histopathological changes in the uterus were detected by HE staining. Based on the oxytocin-induced isolated uterine contraction model, the effects of Gegen Decoction, PLR-free Gegen Decoction, and PLR on the amplitude, frequency, and activity of isolated uterine contraction were compared to investigate the role of PLR in Gegen Decoction for the treatment of PD. The results showed that compared with the Gegen Decoction group, the PLR-free Gegen Decoction improved the indicators of PD except for E_2 content, ER_α expression, and uterine artery blood flow. PLR could significantly down-regulate the serum content of E_2 and the protein expression of uterine ER_α, and improve the uterine artery blood flow. The data suggested that PLR, as the sovereign drug of Gegen Decoction, might function in Gegen Decoction for the treatment of PD by mediating E_(2 )and improving the uterine artery blood flow.