Accurate receptor/ligand binding free energy calculations can greatly accelerate drug discovery by identifying highly potent ligands. By simulating the change from one compound structure to another, the relative binding free energy (RBFE) change can be calculated based on the theoretically rigorous free energy perturbation (FEP) method. However, existing FEP-RBFE approaches may face convergence challenges due to difficulties in simulating non-physical intermediate states, which can lead to increased computational costs to obtain the converged results. To fundamentally overcome these issues and accelerate drug discovery, a new combined-structure RBFE (CS-FEP) calculation strategy was proposed, which solved the existing issues by constructing a new alchemical pathway, smoothed the alchemical transformation, increased the phase-space overlap between adjacent states, and thus significantly increased the convergence and accelerated the relative binding free energy calculations. This method was extensively tested in a practical drug discovery effort by targeting phosphodiesterase-1 (PDE1). Starting from a PDE1 inhibitor (compound 9, IC₅₀ = 16.8 μmol/L), the CS-FEP guided hit-to-lead optimizations resulted in a promising lead (11b and its mesylate salt formulation 11b-Mesylate, IC₅₀ = 7.0 nmol/L), with ∼2400-fold improved inhibitory activity. Further experimental studies revealed that the lead showed reasonable metabolic stability and significant anti-fibrotic effects in vivo.

Objective:A rare case of δ-globin gene (HBD) mutation in Guangdong Province was analyzed to provide reference for avoiding misdiagnosis of δ-thalassemia in clinic.Methods:The patient was admitted to Guangdong Maternal and Child Health Hospital, and the peripheral blood sample was collected for hematological phenotypes [mean erythrocyte volume (MCV), mean erythrocyte hemoglobin content (MCH), hemoglobin (Hb)] and Hb typing analysis. The routine deletion and mutation of α-thalassemia and β-thalassemia genes were analyzed by PCR-flow fluorescence hybridization. At the same time, DNA sequencing was used to analyze the type of HBD mutation.Results:The results of hematological phenotypes analysis showed that MCV was 87.9 fl, MCH was 29.3 pg, and Hb content was 140 g/L. The results of Hb typing showed that the contents of Hb F, Hb A 2, Hb A 2 variant, and Hb A were 0.4%, 1.3%, 0.6%, and 97.7%, respectively. No abnormality was found in α-thalassemia and β-thalassemia genes by routine deletion and mutation detection. According to DNA sequencing analysis, the patient had HBD: c.349 C>G variant. Conclusion:The low Hb A 2 content (reference value is 2.5% - 3.5%) in this case is due to the mutation of HBD, HBD: c.349 C>G variant is rare in Chinese population.

This study explored the mechanism of Shenling Baizhu Powder(SLBZP) in the prevention and treatment of type 2 diabetes from the perspective of flora disorder and chronic inflammation. Fifty rats were randomly divided into normal control group, model control group, low-dose SLBZP group, medium-dose SLBZP group, and high-dose SLBZP group, with 10 rats in each group. The rats of 5 weeks old were administrated by gavage with ultrapure water and different doses of SLBZP decoction. The basic indicators such as body weight and blood glucose were monitored every week, and stool and intestinal contents were collected from the rats of 9 weeks old for 16 S rRNA sequencing and metabolomic analysis. An automatic biochemical analyzer was used to measure the serum biochemical indicators, ELISA to measure serum insulin, and chipsets to measure leptin and inflammatory cytokines. The results showed that SLBZP reduced the body weight as well as blood glucose, glycosylated hemoglobin, and lipid levels. In the rats of 9 weeks, the relative abundance of Anaerostipes, Turicibacter, Bilophila, Ochrobactrum, Acinetobacter, and Prevotella decreased significantly in the model control group, which can be increased in the high-dose SLBZP group; the relative abundance of Psychrobacter, Lactobacillus, Roseburia and Staphylococcus significantly increased in the model control group, which can be down-regulated in the high-dose SLBZP group. The differential metabolites of intestinal flora included 4-hydroxyphenylpyruvic acid, phenylpyruvic acid, octanoic acid, 3-indolepropionic acid, oxoglutaric acid, malonic acid, 3-methyl-2-oxovaleric acid, and methylmalonic acid. Moreover, SLBZP significantly lowered the levels of free insulin, insulin resistance and leptin resistance in rats. The variations in the serum levels of interleukin 1β(IL-1β) and monocyte chemoattractant protein-1(MCP-1) showed that SLBZP could alleviate chronic inflammation in rats. In conclusion, SLBZP can regulate intestinal flora and metabolites and relieve chronic inflammation to control obesity and prevent type 2 diabetes.
Animals ; Diabetes Mellitus, Type 2/drug therapy* ; Gastrointestinal Microbiome ; Inflammation/drug therapy* ; Insulin ; Powders ; Rats

Objective:To observe the effect of Lingshao-Zaoren Decoction on urodynamics and the expression of Piezo1 if overactive bladder (OAB) rats. Methods:Thirty SPF grade female SD rats were randomly divided into blank group, model group, Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups, with 6 rats in each group. The OAB rats were modeled by intraperitoneal injection of Cyclophosphamide. After the successful modeling, Tolterodine control group was given 0.36 mg/kg Tolterodine tartrate, the low-dose and high-dose Lingshao-Zaoren Decoction groups were given 1.59 and 3.18 g/kg Lingshao-Zaoren Mianjian granules by gavage, the blank group and model group were given the same amount of distilled water, once a day for 14 days. After 14 days, the urodynamics of rats in each group were detected. The bladder volume and maximum bladder pressure were observed respectively. The pathological changes of bladder tissue were observed by HE staining. The expression of Piezo1 protein in bladder tissue was detected by immunohistochemistry and Western blot. The expression of Piezo1 mRNA in bladder tissue was detected by qPCR. Results:Compared with the blank group, the body weight, bladder volume and maximum bladder pressure of the model group were significantly reduced ( P<0.01). HE staining result showed that the model group had hyperplasia of urinary tract epithelium, degeneration, necrosis and abscission of epithelial cells, infiltration of a large number of inflammatory cells in stroma, vascular proliferation, thickening of vascular wall, hyperplasia of mucosal smooth muscle, disorder of arrangement, and significant up regulation of Piezo1 protein expression ( P<0.01). Compared with the model group, the weight [(244.83 ± 6.05) g, (233.33 ± 11.76) g vs. (219.00 ± 9.70) g] of rats in the Tolterodine control group and high-dose group of Lingshao-Zaoren Decoction significantly increased ( P<0.01), and the bladder volume [(0.93 ± 0.31) ml, (1.17 ± 0.17) ml, (1.21 ± 0.23) ml vs. (0.50 ± 0.16) ml] and maximum bladder pressure [(42.00 ± 3.03) cmH 2O, (45.83 ± 7.19) cmH 2O, (46.83 ± 8.23) cmH 2O vs. (30.50 ± 5.47) cmH 2O] of rats in the Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups were significantly increased ( P<0.01); the bladder epithelial hyperplasia and degeneration degree, interstitial inflammatory cell infiltration degree and vascular hyperplasia degree of rats in the Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups significantly increased. The expression of Piezo1 mRNA (1.50 ± 0.04, 2.05 ± 0.08, 1.44 ± 0.10 vs. 2.56 ± 0.11) and protein in the Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups were significantly decreased ( P<0.01). Conclusion:Lingshao-Zaoren Decoction can increase the bladder volume and maximum bladder pressure of urinary incontinence caused by detrusor overactivity in rats with overactive bladder, which may be related to reduction of Piezo1 expression.

Objective:To explore the therapeutic effect and mechanism of Chaihu Yueju decoction on model rats induced by chronic unpredictable mild stress （CUMS）. Method:The 60 SD rats were randomly divided into normal group， model group， Chaihu Yueju decoction low， medium and high-dose groups（0.3， 0.6， 1.2 g·kg^-1） and fluoxetine hydrochloride group（0.2 mg·kg^-1）， 10 for each group. The model rats was established by chronic unpredictable mild stress for 5 weeks， and then Chaihu Yueju decoction and fluoxetine hydrochloride were given to the corresponding treatment group by gavage for 3 weeks. In the last week of gavage， Morris water maze training and testing were conducted. After the last day of gavage， sugar water preference and other behavioral experiment were tested. The sugar water preference test was used to detect the degree of pleasure deficiency in rats before and after treatment， the open field test was used to detect the depression of rats before and after treatment， the spatial memory ability was tested by Morris water maze. Western blot was used to detect the levels of tumor necrosis factor-α（TNF-α）， 5-hydroxytryptamine receptor 1A （5-HT1A）， extracellular signal-regulated kinase （ERK） and synapse associated protein in hippocampus of each group， Gloji and Nissl staining was used to observe the changes of dendritic spines and Nissl bodies in the hippocampus CA3. Result:Compared with normal group， the weight， sugar water preference rate， the scores of horizontal and vertical movement were significantly reduced （P<0.05）， and the immobility time was significantly prolonged in model group （P<0.05）. Meanwhile， the ability of learning and memory in model group decreased （P<0.05）. Western blot results showed that the levels of TNF-α was significantly increased （P<0.05）， and the levels of 5-HT1A， p-ERK， cyclic adenosine phosphate reactive element binding protein （CREB）， p-CREB， Synapsin-1，Synaptophysin， glutamate receptor subtype-1（GluR-1）and postsynaptic membrane protein-95（PSD-95） in model group were significantly decreased （P<0.05）. Gloji and Nissl staining results showed that the density of dendritic spines and the number of Nissl body in the hippocampal CA3 of the model group was obviously attenuate （P<0.05）. The results show that the neurons were evidently damaged. Compared with model group， the weight， sugar water preference rate， the scores of horizontal and vertical movement were clearly increased （P<0.05） and the immobility time was significantly shortened in fluoxetine hydrochloride group， middle dose and high dose Chaihu Yueju decoction group （P<0.05）. Western blot results showed that the levels of synaptophysin， GluR-1 and PSD-95 were significantly increased in fluoxetine hydrochloride group， middle dose and high dose Chaihu Yueju decoction group. The level of TNF-α was significantly decreased （P<0.05）， the levels of 5-HT1A， p-ERK， CREB， p-CREB and Synapsin-1 were remarkably increased （P<0.05）， especially the high dose group of Chaihu Yueju decoction. Gloji and Nissl staining results showed that the density of dendritic spines and the number of Nissl body in the hippocampal CA3 of high dose group were similarly increased in a dose-dependent manner. Conclusion:Chaihu Yueju decoction could improves the weight， the depressive despair， autonomous activity ability and learning and memory ability of DP model rats. Its mechanism is closely related to attenuating the inflammatory reaction and enhancing the levels of 5-HT1A receptor protein， ERK and synapse related protein， then activating 5-HT/CREB and ERK/CREB signaling pathways， increasing the number and distribution of dendritic spines and repairing damaged neurons in the DP model rat's hippocampus.