Integrating Network Pharmacology and Experimental Verification to Investigate the Mechanisms of Shuangshi Tonglin Capsule to Treat Chronic Prostatitis.

BACKGROUND AND OBJECTIVE: Chronic prostatitis (CP) is one of the most common diseases in young and middle-aged men but lacks effective treatment. Shuangshi Tonglin Capsule (SSTLC) is a clinical drug for the treatment of chronic prostatitis. However, the underlying molecular mechanisms of SSTLC in treating CP are still unclear. In this study, we researched the underlying mechanisms of SSTLC in treating chronic prostatitis. METHODS: The ingredients of SSTLC were received from the TCMSP and BATMAN databases, and the CP targets were collected based on GeneCards and OMIM. Then, the PPI network and the "drug-ingredient-target" network map were constructed. GO and KEGG enrichment analyses by using DAVID. Molecular docking was performed by using AutoDock 4.2 and PyMol. And using animal experiments to verify the potential effect of SSTLC in CP. RESULTS: SSTLC contained 10 herbs, 158 chemical ingredients and 277 targets, 2002, diseaserelated targets were obtained. Network analysis outcomes indicated that VEGFA, TNF, MAPK1, EGFR, and MAPK8 are the key targets of SSTLC in treating chronic prostatitis. Furthermore, molecular docking revealed that quercetin, luteolin, and kaempferol exhibited a strong binding effect. Animal experimental indicated that SSTLC can reduce the pathological damage to prostate tissue. And, we found that high-dose SSTLC significantly reduced the level of TNF-α and downregulated the expression of EGFR, p-p38 and p-ERK1/2 (P<0.05). CONCLUSION: This study determined the pharmacological effects of SSTLC and the potential mechanism of action on SSTLC to treat CP, it provides a new idea for traditional Chinese medicine to treat chronic prostatitis.

Quercetin suppresses apoptosis of chondrocytes induced by IL-1ß via inactivation of p38 MAPK signaling pathway.

The objective of the present study was to investigate the effect of quercetin and evaluate its protective effect on articular cartilage in patients with osteoarthritis (OA), by intervening the p38 pathway. The target factors of quercetin protecting articular cartilage in patients with OA were predicted scientifically and analyzed to predict the possible pathways by using network pharmacology. A pathway predicted to be closely associated with osteoarthritis was chosen for experimental verification in in vitro cells. The optimal intervention drug concentrations were selected by the of Cell Cycle Kit-8 assay, osteoarthritis and inflammatory factors relevant to osteoarthritis, interleukin-1ß and tumor necrosis factor-α, were tested by of enzyme-linked immunosorbent assay, and the expression of relevant proteins and mRNA of the p38 signaling pathway was tested by reverse transcription-quantitative PCR and western blotting, following quercetin intervention. It was found that quercetin, at the concentration of 100 umol/l, can decrease inflammatory factors relevant to OA, inhibit the expression of p38, matrix metalloprotease 13 and ADAMTS in the pathway, and promote the expression of collagen Ⅱ. Therefore, it is postulated that quercetin can lower the expression of inflammatory factors in cartilage for the prevention and treatment of OA, and the expression level of relevant factors can be changed positively by blocking the p38 MAPK signaling pathway. Thus, quercetin can promote the repair of degenerative chondrocytes and protect articular chondrocytes.

PbTTG1 forms a ribonucleoprotein complex with polypyrimidine tract-binding protein PbPTB3 to facilitate the long-distance trafficking of PbWoxT1 mRNA.

The grafting of horticultural crops enables breeders to induce phenotypic changes in rootstocks and scions. A number of signaling molecules, including RNAs and proteins, were recently shown to underlie these changes; however, little is known about the composition of ribonucleoprotein (RNP) complexes or how these macromolecules are transported. Here, we used a polypyrimidine tract-binding protein, PbPTB3, as a bait to screen a library of phloem cDNA from a pear variety 'Du Li' (Pyrus betulaefolia). We identified a new protein constituent of the RNP complex, TRANSPARENT TESTA GLABRA1 (PbTTG1), a WD40 protein that interacts with PbPTB3 to facilitate its transport with PbWoxT1 mRNA through the phloem. Overexpression experiments indicated that PbTTG1 binds to PbPTB3, facilitating its transmission from the leaf through the petiole, while silencing of PbTTG1 expression prevented their translocation. Heterografting experiments also showed that silencing of PbTTG1 prevented the transport of PbPTB3 from the rootstock to the scion. Collectively, these findings established that PbTTG1 binds to PbPTB3 and PbWoxT1 to form an RNP complex, which facilitates their long-distance movement.

Cardiovascular disease outcomes in metabolically healthy obesity in communities of Beijing cohort study.

OBJECTIVE: The objective of this study was to investigate the association between metabolically healthy obese (MHO) phenotype and the risk of cardiovascular disease (CVD). METHODS: A total of 9393 subjects aged ≥40 years were enrolled in the cohort study (2011-2015). The participants were stratified by body mass index category and metabolic risk at baseline, and incidence of CVD was ascertained at follow-up. RESULTS: The MHO accounted for 6.7%. Compared with the metabolically healthy normal weight (MHNW) group, MHO subjects demonstrated increased risk of CVD events (HR = 1.91; 95% CI, 1.13-3.24). In people with obesity, there was no significant difference on increasing risk of incidence of CVD in the metabolically unhealthy individuals compared with metabolically healthy individuals (HR = 1.19; 95% CI, 0.74-1.91). Female (OR = 1.97; 95% CI, 1.06-3.64), smoking (OR = 2.09; 95% CI, 1.06-4.10), a larger waist circumference (OR = 1.07; 95% CI, 1.03-1.10) and higher LDL cholesterol levels (OR = 1.55; 95% CI, 1.20-2.00) were independent risk factors of the development of the MHO to the metabolically unhealthy obese (MUO) phenotype. CONCLUSIONS: The risk of CVD events of MHO phenotypes is similar to MUO phenotypes; both are higher than the MHNW phenotypes.

Multi-genic pattern found in rare type of hypopituitarism: a whole-exome sequencing study of Han Chinese with pituitary stalk interruption syndrome.

Pituitary stalk interruption syndrome (PSIS) is a rare type of hypopituitarism manifesting various degrees of pituitary hormone deficiency. Although mutations have been identified in some familial cases, the underpinning mechanisms of sporadic patients with PSIS who are in a vast majority remain elusive, necessitating a comprehensive study using systemic approaches. We postulate that other genetic mechanisms may be responsible for the sporadic PSIS. To test this hypothesis, we conducted a study in 24 patients with PSIS of Han Chinese with no family history using whole-exome sequencing (WES) and bioinformatic analysis. We identified a group of heterozygous mutations in 92% (22 of 24) of the patients, and these genes are mostly associated with Notch, Shh, Wnt signalling pathways. Importantly, 83% (20 of 24) of the patients had more than one mutation in those pathways suggesting synergy of compound mutations underpin the pathogenesis of sporadic PSIS.

Exendin-4 Upregulates Adiponectin Level in Adipocytes via Sirt1/Foxo-1 Signaling Pathway.

Glucagon-like peptide-1 (GLP-1) receptor plays an essential role in regulating glucose metabolism. GLP-1 receptor agonists have been widely used for treating diabetes and other insulin resistance-related diseases. However, mechanisms underlying the anti-diabetic effects of GLP-1 receptor agonists remain largely unknown. In this study, we investigated the effects of GLP-1 agonist exendin-4 on the expression of adiponectin, an insulin sensitizing hormone. We found that exendin-4 increased the expression and secretion of adiponectin both in vitro and in vivo. Our data showed that exendin-4 upregulated adiponectin expression at both mRNA and protein levels in adipocytes and adipose tissues. The effects of exendin-4 on adiponectin expression were dependent on the GLP-1 receptor. We further demonstrated important roles of Sirt1 and transcriptional factor Foxo-1 in mediating the function of exendin-4 in regulating adiponectin expression. Suppression of Sirt1 or Foxo-1 expression significantly impaired exendin-4-induced adiponectin expression. Consistently, exendin-4 up-regulated Sirt1 and Foxo-1 expression in vivo. Our work is the first study demonstrating the role of Sirt1/Foxo-1 in regulating the regulatory function of a GLP-1 receptor agonist in adiponectin expression both in vitro and in vivo. The results provide important information for the mechanism underlying the function of GLP-1R on improving insulin resistance and related diseases.

Rapid Pore Cranial Drilling With External Ventricular Drainage for Treatment of Intraventricular Hemorrhage: A 36-Year Case Series.

This study aimed to describe the technique details of rapid pore cranial drilling with external ventricular drainage and document its clinical outcomes by highlighting the advantages over the traditional and modified cranial drilling technique. Intraventricular hemorrhage is one of the most severe subtypes of hemorrhagic stroke with high mortality. The amount of blood in the ventricles is associated with severity of outcomes, and fast removal of the blood clot is the key to a good prognosis. Between 1977 and 2013, 3773 patients admitted for intraventricular hemorrhage underwent rapid pore cranial drilling drainage. The therapeutic effects and clinical outcomes were retrospectively analyzed. Of these patients, 1049 (27.8%) experienced complete remission, 1788 (47.4%) had improved condition, and 936 (24.8%) died. A total of 3229 (85.6%) patients gained immediate remission. One typical case was illustrated to demonstrate the efficacy of the rapid pore drilling technique. Rapid pore cranial drilling drainage in patients with intraventricular hemorrhage is fast, effective, and provides immediate relief in patients with severe conditions. It could be a better alternative to the conventional drilling approach for treatment of intraventricular hemorrhage. A randomized controlled trial for direct comparison between the rapid pore cranial drilling drainage and conventional drilling technique is in urgent need.

Palmitate induces autophagy in pancreatic ß-cells via endoplasmic reticulum stress and its downstream JNK pathway.

Endoplasmic reticulum (ER) stress and autophagy have both been reported to be associated with lipotoxicity in ß-cells, yet the relationship between them has not been fully clarified. In the present study, we tested the hypothesis that the ER stress-autophagic pathway in ß-cells is a downstream pathway activated following saturated fatty acid treatment. Mouse insulinoma (MIN6) ß-cells were treated with either palmitate or thapsigargin (TG) with or without various inhibitors. The results indicated that palmitate strongly enhanced the protein expression of microtubule-associated protein 1 light chain 3 (LC3)-II. Furthermore, the expression levels of ER stress markers, BiP and CHOP, and phosphorylation levels of JNK were increased after palmitate treatment. In addition, palmitate-induced autophagy was blocked by 500 µM of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or 20 µM JNK inhibitor SP600125. In turn, the phosphorylation of Akt (Ser473) was also downregulated by palmitate, while the levels of insulin receptor ß (IRß) were not reduced. A further increase in LC3-II levels was observed in cells treated with both palmitate and 50 µM PI3K/Akt inhibitor LY294002 compared with cells treated with palmitate alone. Palmitate-induced phospho-Akt (Ser473) downregulation was also inhibited by TUDCA or SP600125. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA, 5 mM) for 1 h increased the expression of ER stress markers, and enhanced cell injuries caused by 0.1 µM TG, including decreased cell viability and insulin secretion. Palmitate induces autophagy in pancreatic ß-cells possibly through activation of ER stress and its downstream JNK pathway. Palmitate-induced autophagy may protect ß-cells against cell injuries caused by ER stress.

Pituitary stalk interruption syndrome in Chinese people: clinical characteristic analysis of 55 cases.

OBJECTIVE: Pituitary stalk interruption syndrome (PSIS) is characterized by the absence of pituitary stalk, pituitary hypoplasia, and ectopic posterior pituitary. Due to the rarity of PSIS, clinical data are limited, especially in Chinese people. Herein, we analyzed the clinical characteristics of patients diagnosed with PSIS from our center over 10 years. PATIENTS AND METHODS: We retrospectively analyzed the clinical manifestations and laboratory and MRI findings in 55 patients with PSIS. RESULTS: Of the 55 patients with PSIS, 48 (87.3%) were male. The average age was 19.7±6.7 years and there was no familial case. A history of breech delivery was documented in 40 of 45 patients (88.9%) and 19 of 55 patients (34.5%) had a history of dystocia. Short stature was found in 47 of 55 patients (85.5%) and bone age delayed 7.26±5.37 years. Secondary sex characteristics were poor or undeveloped in most patients. The prevalence of deficiencies in growth hormone, gonadotropins, corticotropin, and thyrotropin were 100%, 95.8%, 81.8%, 76.3%, respectively. Hyperprolactinemia was found in 36.4% of patients. Three or more pituitary hormone deficiencies were found in 92.7% of the patients. All patients had normal posterior pituitary function and absent pituitary stalk on imaging. The average height of anterior pituitary was 28 mm, documented anterior pituitary hypoplasia. Midline abnormalities were presented in 9.1% of patients. CONCLUSIONS: The clinical features of our Chinese PSIS patients seem to be different from other reported patients in regarding to the higher degree of hypopituitarism and lower prevalence of midline defects. In addition, our patients were older at the time of case detection and the bone age was markedly delayed. We also had no cases of familial PSIS.

Pituitary stalk interruption syndrome in 58 Chinese patients: clinical features and genetic analysis.

OBJECTIVES: Pituitary stalk interruption syndrome (PSIS) is rare and its clinical features and pathogenesis are poorly understood. This study characterized the clinical and genetic features of PSIS in Chinese patients. DESIGN AND PATIENTS: Clinical data of 58 patients with PSIS and 46 patients with GH deficiency but a normal pituitary stalk (NPS) were retrospectively analysed. HESX1, LHX4, OTX2 and SOX3 polymorphisms were screened in 33 PSIS patients, and GH1 and GHRHR in 4 NPS patients. RESULTS: Deficiency of GH was 100% in both PSIS and NPS groups. Other deficiency rates for PSIS and NPS groups were as follows: ACTH, 77·6% and 23·9%; TSH, 43·1% and 10·9%; LH/FSH, 94·2% and 47·4%; and combined pituitary hormone, 93·1% and 41·3% respectively. In PSIS and NPS patients, the percentages of anterior pituitary hypoplasia were 98·3% and 54·3%, pituitary stalk abnormality were 100% and 0%, and ectopic neurohypophysis were 91·4% and 0%. A novel heterozygous sequence variant (c.142A>T, p.T48S) was found in HESX1 in one PSIS patient, 3 polymorphisms (c.63T>C, p.G21G; c.450C>T, p.N150N; and c.983A>G, p.N328S) in LHX4 in 7, 1 and 31 PSIS patients, respectively, and a hemizygous polymorphism (c.157G>C, p.V53L) in SOX3 in one PSIS patient. No OTX2 abnormality was detected in PSIS patients, and no GH1 or GHRHR polymorphisms in NPS patients. CONCLUSIONS: Compared with NPS, PSIS patients had more severe anterior pituitary hormone deficiency, lower anterior pituitary hormone secretion and higher probability of abnormal pituitary morphology. HESX1, LHX4 and SOX3 polymorphisms may be associated with PSIS.

[Detrimental effects of homocysteine on insulin release and apoptosis of pancreatic beta cells].

OBJECTIVE: To study the effects of homocysteine (Hcy) on the insulin secretion and apoptosis of pancreatic beta cells. METHODS: Clonal mouse pancreatic beta cells of the line NIT-1 were cultured and exposed to Hcy of 50, 100, 250, 500 and 1000 micromol/L for 6, 12, 24, and 48 hours respectively, then glucose of the concentrations of 5.6 mmol/L or 16.7 mmol/L was added for 1 h, and then the insulin concentration in the culture medium was determined by radioimmunoassay, and MTT method was used to detect the survival rate of the cells. NIT-1 cells were exposed to Hcy of the concentrations of 0, 50, 100, 250, 500 and 1000 micromol/L respectively for 24 h, another NIT-1 cells were exposed to Hcy of the final concentration of 250 micromol/L for 0, 6, 12, and 48 h respectively, then flow cytometry (FC) was used to detect the apoptosis of the cells. After exposure to Hcy of the concentrations of 50, 100, 250, 500 and 1000 micromol/L for 72 h DNA agarose gel electrophoresis was performed. RESULTS: Hcy inhibited the basal and glucose-induced insulin secretion in a time- and dose-dependent manner. The insulin secretion amounts of the NIT-1 cells after exposure to 50 micromol/L Hcy for 24 hours and to 100 micromol/L Hcy for 12 hours were significantly lower by 17.1% and 10.8% compared with the control group (all P < 0.01). Incubated with 100 micromol/L Hcy for 12 hours and 24 hours respectively, the survival rates of the NIT-1 cells were 94.56% and 87.93% respectively (P < 0.05, P < 0.01). Incubated with 100 micromol/L Hcy for 24 hours, the apoptosis rate of the NIT-1 cells was 7.21% (P < 0.01); and incubated with 250 micromol/L Hcy for 12 hours, the apoptosis rate of the NIT-1 cells was 12.93% (P < 0.01). Both FC and agarose gel electrophoresis indicated that Hcy induced cell apoptosis time- and concentration-dependently. CONCLUSION: Hcy impairs insulin secretion and induces cell apoptosis of pancreatic beta cells time- and concentration-dependently.

Two novel mutations 685del 1 and D129G in the low-density lipoprotein receptor gene in a compound heterozygote Chinese family with familial hypercholesterolemia.

DNA sequencing analysis was used to scan the genes in a Chinese family with clinically diagnosed autosomal genetic hypercholesterolemia. Two mutations were identified in exon 4 of the low-density lipoprotein receptor gene, which is the possible molecular mechanism of etiology of the family. The proband's extremely high level of serum cholesterol and the related manifestations suggested that he was a familial hypercholesterolemia homozygote and that his parents were in a relatively milder condition. DNA sequencing revealed that the proband had an abnormal pattern of exon 4 of the low-density lipoprotein receptor gene due to a heterozygosity (A/G) at nucleotide 386 and a heterozygous single-base deletion (A) at 685. Nucleotide 386 is the second base of codon 129, and A-->G mutation (D129G) changed this codon from Asp(GAC) to Gly(GGC). The single-base deletion of A at 685 (685del 1) is a frameshift mutation. It changes the phase of triplets, so that all codons are misread after this site of mutation; consequently, the protein expressed by the gene must be abnormal in structure and function. DNA analysis of the other family members showed that the 2 mutations should be respectively located in different alleles of the proband. Both of the 2 mutations have not been reported previously.

[A compound heterozygote family of familial hypercholesterolemia due to new mutations].

OBJECTIVE: To investigate the molecular diagnosis method and possible molecular mechanism of the etiology of a hereditary genetic hypercholesterolemia family by scanning and analyzing the related genes of hereditary hypercholesterolemia in a clinically diagnosed proband and his family members. METHODS: Molecular diagnosis was performed with PCR and then DNA sequencing of the promoter and 18 exons of low-density lipoprotein receptor (LDLR) gene and 3500 - 3531 fragment of apolipoprotein B-100 gene was carried out. The sequencing results were compared with the normal nucleotide sequence queried from the GeneBank database to discover the mutations. RESULTS: Familial defective apolipoprotein B-100 was excluded, as no mutation was detected in the apolipoprotein B 3500 - 3531 fragment. Two new point mutations were detected in the exon 4 of the proband's LDLR gene, they were heterozygous 685delA (Del A at 685) and 386A > G. The sequencing in his parents and other family members showed that the two mutations were paternal origin (685delA) and maternal origin (386A > G) respectively and should be located in different alleles of the proband. CONCLUSION: Molecular diagnosis in the family shows that the proband is a compound heterozygote and the newly detected LDLR gene mutations of 685delA and 386A > G are the possible molecular etiological basis of the disease in this family.

[Improving the affinity of an anti-TNF-alpha scFv by random mutation].

AIM: To improve the affinity of an anti-TNF-alpha scFv. METHODS: A mutant phage antibody library derived from an anti-TNF-alpha scFv gene was generated by error-prone PCR. The mutated genes were then subjected to DNA shuffling. Mutants with improved affinity were selected by bio-panning. Affinity improvement of the selected mutants was verified by dot blot ELISA and thiocyanate elusion ELISA. RESULTS: One mutant was obtained with relative affinity index (1.37 mol/L) higher than that of the parent scFv (0.48 mol/L). CONCLUSION: Error-prone PCR plus DNA shuffling is effective in improving the affinity of antibodies.

Saturated free fatty acids, palmitic acid and stearic acid, induce apoptosis by stimulation of ceramide generation in rat testicular Leydig cell.

In men, obesity has generally been associated with reduced plasma testosterone levels and with elevation of the plasma free fatty acids (FFAs). In this study, we investigated the effects of saturated FFAs including palmitic acid (PA) and stearic acid (SA), and polyunsaturated FFA arachidonic acid (AA) on the survival of rat testicular Leydig cell cultured in vitro. PA and SA markedly suppressed Leydig cell survival in a time- and dose-dependent manner. In contrast, AA stimulated the cell proliferation at 5-10 times of physiological concentration. The suppressive effect of PA and SA on cell survival was caused by apoptosis evidenced by DNA ladder formation and Annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effect of PA was possibly mediated by ceramide generation because it could be completely blocked by ceramide synthase inhibitor fumonisin B1 and exogenous ceramide itself could directly induce apoptosis in vitro. Surprisingly, the apoptosis induced by PA could be partly prevented by AA. These results indicate that PA and SA induce apoptosis in testicular Leydig cells by ceramide production and these apoptotic effects may be a possible mechanism for reproductive abnormalities in obese men, and AA can partly prevent the apoptotic effect induced by saturated FFA.

[Establishment of drug-resistance cell line of human glioma mediated by MGMT].

OBJECTIVE: To establish a drug-resistance cell line of human glioma mediated by MGMT. METHODS: Simulated the clinical usage of BCNU to establish a BCNU-resistant human glioma subline by cyclic exposing the U251 parent cells to a constant concentration of BCNU. The resistance index and the expression of MGMT mRNA of U251/BCNU were detected and compared the difference of in vitro proliferation between U251 and U251/BCNU. RESULTS: A subline--U251/BCNU was successfully established in about 4-month culture, which had a stable resistance to BCNU. U251/BCNU cells showed 17-fold higher resistance to BCNU than did U251 cells by MTT assay, while U251/BCNU cells expressed MGMT mRNA. The doubling time of U251 and U251/BCNU had no statistical difference. CONCLUSION: A drug-resistance cell line of human glioma mediated by MGMT is established, which could provide experimental basis for further studies on the resistance mechanism and reversal methods of glioma chemotherapy.