Generic and accurate prediction of retention times in liquid chromatography by post-projection calibration.

Retention time predictions from molecule structures in liquid chromatography (LC) are increasingly used in MS-based targeted and untargeted analyses, providing supplementary evidence for molecule annotation and reducing experimental measurements. Nevertheless, different LC setups (e.g., differences in gradient, column, and/or mobile phase) give rise to many prediction models that can only accurately predict retention times for a specific chromatographic method (CM). Here, a generic and accurate method is present to predict retention times across different CMs, by introducing the concept of post-projection calibration. This concept builds on the direct projections of retention times between different CMs and uses 35 external calibrants to eliminate the impact of LC setups on projection accuracy. Results showed that post-projection calibration consistently achieved a median projection error below 3.2% of the elution time. The ranking results of putative candidates reached similar levels among different CMs. This work opens up broad possibilities for coordinating retention times between different laboratories and developing extensive retention databases.

[Screening of organophosphorus flame retardant residues in rice by QuEChERS-gas chromatography-quadrupole time-of-flight mass spectrometry].

Organophosphorus flame retardants (OPFRs) have emerged as good alternatives to brominated flame retardants, the use of which is globally restricted. In this study, a screening method based on QuEChERS-gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS) was established for the determination of 21 OPFRs in rice. First, full scan (scanning range, m/z 50-450) was performed with a mixed standard solution of the 21 OPFRs (0.1 µg/g) by GC-Q-TOF/MS. The fragmentation pathways of these OPFRs were then investigated to explore their cleavage fragments, the interrelationships among fragments, and the possible cleavage modes of alkylated, chlorinated, and aromatic OPFRs. The retention times, isotopic abundance ratios, and molecular formulas of the characteristic fragments as well as the exact mass of the compounds were obtained to establish a mass spectral library of the OPFRs. Rice samples were extracted and purified by the QuEChERS method, and 0.5% formate acetonitrile solution was used as the extraction solvent; 4 g of magnesium sulfate, 1 g of sodium chloride, 0.5 g of disodium hydrogen citrate, and 1 g of sodium citrate as the extraction-salt combination; and 50 mg of primary secondary amine (PSA), 50 mg of octadecylsilane (C18), and 150 mg of magnesium sulfate as the purification materials. The chromatographic separation of the 21 OPFRs was completed within 16 min under optimized temperature program conditions on the DB-5MS UI column. The screening parameters were optimized, and a full scan of the samples was performed under the following conditions: number of characteristic fragment ions ≥2; accurate mass window=±2×10-5 (±20 ppm); retention time deviation=±0.2 min, and ion abundance deviation<20%. The developed method was applied to the screening 21 OPFRs in the samples. The results indicated that the matrix interference was greatly reduced by decreasing the extraction accurate mass window, thereby improving the signal-to-noise ratio of the analytes. The targets were extracted from the matrix interference and background noise using deconvolution software, which improved the match between the target compounds and the mass spectral library. The detection rates of alkyl and aromatic OPFRs increased by 22% and 25%, respectively, when the spiking level was increased from 2 to 10 ng/g. Among the chlorinated OPFRs, only tris(2-chloroisopropyl) phosphate (TCIPP) was not detected at a spiking level of 2 ng/g, indicating that chlorinated OPFRs could be identified even at low concentrations. The characteristic ions of the detected compounds matched those of the home-made mass spectral library well, indicating that the practical application of the home-made mass spectral library. The established screening method was applied in the determination of OPFRs in rice samples from different regions in China. A total of 11 OPFRs were detected, among which trimethyl phosphate (TMP), tri-iso-butyl phosphate (TiBP), and tris(3,5-dimethylphenyl) phosphate (T35DMPP) had the highest detection rates. These results indicate that these three OPFRs are widely used and can easily come into contact with rice samples through various routes. Differences in the types of OPFRs detected in the actual samples may be related to the types of OPFRs produced in local factories. OPFRs can be detected in rice samples by the developed GC-Q-TOF/MS screening method, which is helpful for the identification of OPFRs in complex matrix samples.

Effect of Kangfuxiaomi suppository on pelvic inflammatory disease in rats.

Pelvic inflammatory disease (PID) is commonly encountered in gynecological practice. Kangfuxiaomi suppository, made from the compound extract of Periplaneta Americana, is a Traditional Chinese Medicine remedy widely used for the treatment of gynecological disorders. This study aimed to preliminarily explore the therapeutic effect of Kangfuxiaomi suppository in a rat model of PID established by chemical injury and pathogen infection. The key parameters assessed were vulvar inflammation score, vaginal + uterine organ index, and serum levels of interleukin (IL)- 8; tumor necrosis factor (TNF)-α; C-reactive protein (CRP); superoxide dismutase (SOD); and malondialdehyde (MDA). In addition, levels of IL-6, cyclooxygenase (COX)- 2, and IL-2 in cervical tissues as well as that of IL-1ß and prostaglandin E-2 (PGE2) in uterine tissues were measured. The expression levels of nuclear factor-kappa B (NF-κB) p65 and Toll-like receptor 4 (TLR4) in uterine tissues were detected by immunohistochemical method. After Kangfuxiaomi suppository treatment, the vulva inflammation score and histopathological score of PID rats showed a tendency to decrease. Serum IL-8, TNF-α, CRP, and MDA levels were reduced, while SOD levels were significantly increased. Levels of IL-6, IL-2, and COX-2 in cervical tissues were somewhat decreased, and PGE2 and IL-1ß levels in uterine tissue were significantly decreased. Moreover, the levels of NF-κB p65 and TLR4 protein expression were also decreased. These findings demonstrated the therapeutic effect of Kangfuxiaomi suppository in PID rats. The underlying mechanism may involve enhanced antioxidant capacity and decreased secretion of proinflammatory factors via the NF-κB/TLR4 signaling pathway.

[A Prediction Model for Human Immunodeficiency Virus Infection and Mother-to-Child Transmission Based on the Expression Levels of Selenoprotein Genes].

Objective To study the expression of selenoprotein genes in human immunodeficiency virus(HIV)infection and its mother-to-child transmission,so as to provide a theoretical basis for the prevention,diagnosis,and treatment of acquired immunodeficiency syndrome.Methods The dataset GSE4124 was downloaded from the Gene Expression Omnibus(GEO).Two groups of HIV-positive mothers(n=25)and HIV-negative mothers(n=20)were designed.HIV-positive mothers included a subset of transmitter(TR)mothers(n=11)and non-transmitter(NTR)mothers(n=14).Then,t-test was carried out to compare the expression levels of selenoprotein genes between the four groups(HIV-positive vs. HIV-negative,NTR vs. HIV-negative,TR vs. HIV-negative,TR vs. NTR).Univariate and multivariate Logistic regression were adopted to analyze the effects of differentially expressed genes on HIV infection and mother-to-child transmission.R software was used to establish a nomogram prediction model and evaluate the model performance.Results Compared with the HIV-negative group,HIV-positive,NTR,and TR groups had 8,5 and 8 down-regulated selenoprotein genes,respectively.Compared with the NTR group,the TR group had 4 down-regulated selenoprotein genes.Univariate Logistic regression analysis showed that abnormally high expression of GPX1,GPX3,GPX4,TXNRD1,TXNRD3,and SEPHS2 affected HIV infection and had no effect on mother-to-child transmission.The multivariate Logistic regression analysis showed that the abnormally high expression of TXNRD3(OR=0.032,95%CI=0.002-0.607,P=0.022)was positively correlated with HIV infection.As for the nomogram prediction model,the area under the receiver-operating characteristic curve for 1-year survival of HIV-infected patients was 0.840(95%CI=0.690-1.000),and that for 3-year survival of HIV-infected patients was 0.870(95%CI=0.730-1.000).Conclusions Multiple selenoprotein genes with down-regulated expression levels were involved in the regulation of HIV infection and mother-to-child transmission.The abnormal high expression of TXNRD3 was positively correlated with HIV infection.The findings provide new ideas for the prevention,diagnosis,and treatment of acquired immunodeficiency syndrome.

ß-arrestin2 deficiency ameliorates S-100-induced autoimmune hepatitis in mice by inhibiting infiltration of monocyte-derived macrophage and attenuating hepatocyte apoptosis.

Autoimmune hepatitis (AIH) is a progressive hepatitis syndrome characterized by high transaminase levels, interface hepatitis, hypergammaglobulinemia, and the presence of autoantibodies. Misdiagnosis or delayed treatment of AIH can lead to cirrhosis or liver failure, which poses a major risk to human health. ß-Arrestin2, a key scaffold protein for intracellular signaling pathways, has been found to be involved in many autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. However, whether ß-arrestin2 plays a role in AIH remains unknown. In the present study, S-100-induced AIH was established in both wild-type mice and ß-arrestin2 knockout (Arrb2 KO) mice, and the experiments identified that liver ß-arrestin2 expression was gradually increased, and positively correlated to serum ANA, ALT and AST levels during AIH progression. Furthermore, ß-arrestin2 deficiency ameliorated hepatic pathological damage, decreased serum autoantibody and inflammatory cytokine levels. ß-arrestin2 deficiency also inhibited hepatocyte apoptosis and prevented the infiltration of monocyte-derived macrophages into the damaged liver. In vitro experiments revealed that ß-arrestin2 knockdown suppressed the migration and differentiation of THP-1 cells, whereas ß-arrestin2 overexpression promoted the migration of THP-1 cells, which was regulated by the activation of the ERK and p38 MAPK pathways. In addition, ß-arrestin2 deficiency attenuated TNF-α-induced primary hepatocyte apoptosis by activating the Akt/GSK-3ß pathway. These results suggest that ß-arrestin2 deficiency ameliorates AIH by inhibiting the migration and differentiation of monocytes, decreasing the infiltration of monocyte-derived macrophages into the liver, thereby reducing inflammatory cytokines-induced hepatocytes apoptosis. Therefore, ß-arrestin2 may act as an effective therapeutic target for AIH.

CP-25 exerts a protective effect against ConA-induced hepatitis via regulating inflammation and immune response.

Hepatitis is a complex multifactorial pathological disorder, which can eventually lead to liver failure and even potentially be life threatening. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has proven to have critical anti-inflammatory effects in arthritis. However, the effects of CP-25 in the pathogenesis of hepatitis remains unclear. In this experiment, mice were intragastrically administered with CP-25 (25, 50 and 100 mg/kg), and then ConA (25 mg/kg) was intravenous injected to establish hepatitis model in vivo. CP-25 administration attenuated liver damage and decreased ALT and AST activities in mice with hepatitis. Besides, CP-25 modulated immune responses including down-regulated the proportions of activated CD4+, activated CD8+ T cells, and ratio of Th1/Th2 in ConA-injected mice. Furthermore, ConA-mediated production of reactive oxygen species (ROS), release of inflammatory cytokines including IFN-γ, TNF-α, activation of MAPK pathways and nuclear translocation of nuclear factor-kappaB (NF-κB) were significantly decreased in CP-25 administrated mice. In ConA-stimulated RAW264.7 cells, CP-25 suppressed inflammatory cytokines secretion and reduced ROS level, which were consistent with animal experiments. Otherwise, the data showed that CP-25 restrained phosphorylation of ERK, JNK and p38 MAPK pathways influenced by ROS, accompanied with inhibiting NF-κB nuclear translocation. In conclusion, our findings indicated that CP-25 protected against ConA-induced hepatitis may through modulating immune responses and attenuating ROS-mediated inflammation via the MAPK/NF-κB signaling pathway.

Pre-diagnostic dietary consumption of calcium and magnesium and calcium-to-magnesium intake ratio and ovarian cancer mortality: results from the ovarian cancer follow-up study (OOPS).

PURPOSE: Previous studies have indicated that dietary consumption of calcium (Ca), magnesium (Mg), and the Ca-to-Mg (Ca:Mg) ratio were associated with different health outcomes. However, no study has evaluated the association of pre-diagnostic Ca, Mg, and Ca:Mg ratio consumption with ovarian cancer (OC) survival. METHODS: The aforementioned associations were investigated in a cohort of 853 Chinese women diagnosed with OC between 2015 and 2020. A validated food frequency questionnaire was used to evaluate pre-diagnostic diet information. Deaths were recorded until March 31, 2021 via medical records and active follow-up. Cox proportional hazards model was applied to calculate the adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: A total of 130 deaths were observed during a median follow-up of 37.2 months. After adjustment for potential confounders, pre-diagnostic Ca (HR< 600 vs. > 1000 = 1.45, 95% CI = 0.47-4.46, p for trend = 0.69) and Mg (HR< 250 vs. > 330 = 0.90, 95% CI = 0.39-2.08, p for trend = 0.77) intakes were found to be unrelated to OC survival, whereas a higher Ca:Mg intake ratio was significantly associated with worse survival (HR< 1.7 vs. > 2.5 = 2.72, 95% CI = 1.28-5.78, p for trend < 0.05). A significant result was also observed when treating the Ca:Mg ratio as a continuous variable (HR = 1.69, 95% CI = 1.12-2.55) for one-unit increment. CONCLUSION: Pre-diagnostic consumption of Ca and Mg was unrelated to OC survival, while a higher Ca:Mg intake ratio was strongly associated with worse survival among OC patients.

Pre-diagnosis meat intake and cooking method and ovarian cancer survival: results from the Ovarian Cancer Follow-Up Study (OOPS).

Objectives: The relationships between pre-diagnosis meat intake and ovarian cancer (OC) survival were limited and controversial. To date, no study has taken account of cooking methods. Thus, we aimed to firstly clarify these associations based on the Ovarian Cancer Follow-Up Study. Methods: This prospective cohort study, including 853 OC patients between 2015 and 2020, was conducted to examine the aforementioned associations. All women completed a food frequency questionnaire. Deaths were ascertained up to March 31, 2021 via medical records and active follow-up. We used the Cox proportional hazards model to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Results: During the median follow-up of 37.17 months, 130 women died. Pre-diagnosis fish and seafood intake was associated with better survival (HRT3 vs. T1 = 0.46, 95% CI = 0.26-0.82, p trend <0.05), whereas processed red meat (HR = 1.54, 95% CI = 1.04-2.26) and a high frequency of fried fish intake (HR = 1.49, 95% CI = 1.03-2.16) were associated with worse survival than consuming none. After considering the interaction of cooking methods, we found that compared with the lowest tertile of fish and seafood intake and almost no fried fish cooking, women with the highest tertile of intake and almost no fried fish cooking had better survival (HR = 0.35, 95% CI = 0.13-0.92). Additionally, compared with the lowest tertile of fish and seafood intake and almost no baked fish cooking, women with the lowest tertile of intake and consuming baked fish had worse survival (HR = 3.75, 95% CI = 1.53-9.15). Conclusions: Pre-diagnosis fish and seafood intake was associated with better OC survival, whereas processed red meat intake was associated with worse survival. Cooking methods, especially for fried or baked fish, may play interaction effects with fish intake on OC survival.

Association between pre-diagnostic dietary pattern and survival of ovarian cancer: Evidence from a prospective cohort study.

BACKGROUND: Evidence for a role of single nutrition or foods on ovarian cancer (OC) survival has been limited and inconclusive. Due to the potentially complex interactions in dietary, we applied dietary patterns to this study to firstly explore the relationship between the pre-diagnosis overall diet and OC survival. METHODS: The prospective cohort study was conducted among 853 OC patients aged 18-79 years during 2015-2020. Dietary intake was collected using a validated 111-item food frequency questionnaire. Deaths were obtained by medical records and cancer registry up to March 31, 2021. Cox proportional hazards regression models was used to evaluate the hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of pre-diagnosis dietary patterns with overall survival (OS). RESULTS: Overall, during the follow-up period (median: 37.57 months, interquartile: 25.00-50.17 months), 130 (18.49%) OC patients died. Four dietary patterns were identified: healthy pattern, ethnic pattern, animal foods pattern, and sweet pattern. The highest tertile of the healthy pattern scores was related to better OS compared with the lowest tertile scores (HR = 0.54, 95% CI = 0.30-0.98, p trend <0.05), whereas OC patients with highest adherence to the animal foods pattern was associated with worse OS than those with the lowest adherence (HR = 1.90, 95% CI = 1.14-3.17, p trend <0.05). We found no significant associations between adherence to ethnic pattern and sweet pattern and OS of OC patients. CONCLUSION: Pre-diagnosis healthy patterns was associated with better OC survival, whereas animal pattern was associated with worse survival among OC survivals.

Pre-diagnosis Cruciferous Vegetables and Isothiocyanates Intake and Ovarian Cancer Survival: A Prospective Cohort Study.

Background: The associations of the consumption of cruciferous vegetables (CVs) and their bioactive components, isothiocyanates (ITCs), with ovarian cancer (OC) mortality have been unclear, owing to limited studies and inconsistent findings. To date, no studies have evaluated these associations among Chinese patients with OC. This study aims to provide more evidence indicating the relationships of pre-diagnosis CVs and ITC intake with OC survival. Methods: We examined the associations of pre-diagnosis CV and ITC intake with OC mortality in a hospital-based cohort (n = 853) of Chinese patients with epithelial OC between 2015 and 2020. Pre-diagnosis dietary information was evaluated with a validated food frequency questionnaire. Deaths were ascertained until March 31, 2021 via medical records and active follow-up. The associations were examined with the Cox proportional hazards model, adjusted for potential confounders, and stratified by menopausal status, residual lesions, histological type, and body mass index (BMI). Results: During a median follow-up of 37.2 months (interquartile: 24.7-50.2 months), we observed 130 deaths. The highest tertile of total CV intake was associated with better survival than the lowest tertile intake [hazard ratio (HR) = 0.57, 95% confidence interval (CI) = 0.33-0.98, p trend < 0.05]. In addition, higher intake of ITCs from CVs was associated with better survival (HRT3VS.T1 = 0.59, 95% CI = 0.36-0.99, p trend = 0.06). Significant inverse associations were also observed for subgroup analyses stratified by menopausal status, residual lesions, histological type, and BMI, although not all associations showed statistical significance. Conclusion: Increasing pre-diagnosis consumption of CVs and ITCs was strongly associated with better survival in patients with OC.

To explore the therapeutic effect of Kangfuxiaomi shuan II on cervicitis in rats.

PURPOSE: The rat cervicitis model was established with 20% phenol glue to explore the therapeutic effect of Kangfuxiaomi shuan II on rat cervicitis and its mechanism. METHODS: After modeling, the rats were treated with Shuangzuotai suppository (37.84 mg/kg), Kangfuxiaoyan shuan (205.6 mg/kg) and Kangfuxiaomi shuan II (40, 80, 160 mg/kg). The histopathological changes and injury degree of cervix in rats were evaluated by vulvar inflammation score and organ index. The therapeutic effect of Kangfuxiaomi shuan II on cervicitis was evaluated by detecting the levels of copper-protein (CP), C-reactive protein (CRP), Rat interleukin 6 (IL-6), superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and epidermal growth factor (EGF), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cervical tissue. RESULTS: Compared with the model group, the vulvar inflammation score and cervical index of rats in other groups decreased significantly (P<0.01). Kangfuxiaomi shuan II could significantly reduce the levels of CP, CRP, and MDA in serum of rats with cervicitis, and significantly increase the activity of SOD in serum of rats with cervicitis (P<0.01). The levels of EGF and iNOS in cervical tissue of rats also increased in different degrees, while the level of COX-2 decreased significantly (P<0.01), which significantly improved the pathological degree of vulvar inflammation in rats with cervicitis. CONCLUSIONS: Kangfuxiaomi shuan II has a certain therapeutic effect on cervicitis in rats, and its mechanism may be related to the regulation of inflammatory cytokine network and immunity.

Pre-diagnosis Dairy Product Intake and Ovarian Cancer Mortality: Results From the Ovarian Cancer Follow-Up Study (OOPS).

Background: Dairy product consumption is associated with ovarian cancer (OC) incidence. However, limited evidence is available on its influence on OC mortality. Methods: The association between pre-diagnostic dairy product intake and OC mortality was investigated in the OC follow-up study, which included a hospital-based cohort (n = 853) of women diagnosed with epithelial OC between 2015 and 2020. Pre-diagnosis diet information was collected using a validated food frequency questionnaire. Deaths were ascertained up to March 31, 2021 via death registry linkage. Cox proportional hazards model was used to estimate the adjusted hazard ratio (HR) and 95% confidence interval (CI) for the aforementioned association. Results: A total of 130 women died during the median follow-up of 37.2 months (interquartile: 24.7-50.2 months). Comparisons of highest to lowest tertile intake showed that pre-diagnosis dairy product use was associated with total OC mortality (HR = 2.03, 95% CI = 1.21-3.40, p trend = 0.06). In addition, short survival was separately associated with protein (HR = 2.09, 95% CI = 1.25-3.49, p trend < 0.05), fat (HR = 2.16, 95% CI = 1.30-3.61, p trend < 0.05), and calcium (HR = 2.03, 95% CI = 1.21-3.4, p trend = 0.06) from dairy intake. Similar positive magnitudes were observed for menopausal status, residual lesions, histological type, and body mass index, although not all of these factors showed statistical significance. Conclusion: Pre-diagnosis dairy product consumption, including protein, fat, and calcium from dairy intake, was associated with higher mortality among OC survivors.

Review of Mendelian Randomization Studies on Ovarian Cancer.

Ovarian cancer (OC) is one of the deadliest gynecological cancers worldwide. Previous observational epidemiological studies have revealed associations between modifiable environmental risk factors and OC risk. However, these studies are prone to confounding, measurement error, and reverse causation, undermining robust causal inference. Mendelian randomization (MR) analysis has been established as a reliable method to investigate the causal relationship between risk factors and diseases using genetic variants to proxy modifiable exposures. Over recent years, MR analysis in OC research has received extensive attention, providing valuable insights into the etiology of OC as well as holding promise for identifying potential therapeutic interventions. This review provides a comprehensive overview of the key principles and assumptions of MR analysis. Published MR studies focusing on the causality between different risk factors and OC risk are summarized, along with comprehensive analysis of the method and its future applications. The results of MR studies on OC showed that higher BMI and height, earlier age at menarche, endometriosis, schizophrenia, and higher circulating ß-carotene and circulating zinc levels are associated with an increased risk of OC. In contrast, polycystic ovary syndrome; vitiligo; higher circulating vitamin D, magnesium, and testosterone levels; and HMG-CoA reductase inhibition are associated with a reduced risk of OC. MR analysis presents a2 valuable approach to understanding the causality between different risk factors and OC after full consideration of its inherent assumptions and limitations.

The identification of fish eggs of two species, the ovate sole Solea ovata and black porgy Acanthopagrus schlegelii.

Fish eggs of the ovate sole Solea ovata and black porgy Acanthopagrus schlegelii were identified through DNA barcoding of cytochrome c oxidase subunit I (COX1). Visual taxonomic features were achieved, and photographs of the eggs of both species at different developmental stages were reported for the first time. In addition, the dissolution of oil globules caused by ethanol as egg fixatives was observed. This result showed the importance of using formalin as egg fixatives in the case of morphometric analysis and the necessity of combining molecular and visual taxonomic method for morphological study.

Assessment of the impact of hydrolysis on bound sulfonamide residue determination in honey using stable isotope dilution ultrahigh performance liquid chromatography tandem mass spectrometry.

In this study, an analytical method based on isotope dilution-liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was developed as a candidate reference method for the determination of sulfonamides (SAs) in honey. To guarantee the accuracy and authenticity, the impact of hydrolysis on bound SA residues was first investigated by enabling (i) identification of sugar-bound SAs, (ii) clarifying the binding reaction rule between the SAs and sugar, (iii) detection of free SAs and sugar-bound SAs, and (iv) preparation of SA-contaminated honey. Thus, the efficiency of different hydrolysis conditions was assessed by comparing the bound SA content before and after hydrolysis. In addition, optimization of the sample pretreatment procedures and LC conditions to minimize matrix effects by separation from significant matrix interferences was also performed. Satisfactory results in terms of hydrolysis efficiency (approximately 88.3%-99.2%), extraction efficiency (84.2%-105.3%), recovery (95.9%-103.1%), and limit of quantification (0.6-1.5 µg·kg-1) were obtained.

G Protein-Coupled Receptor Kinase 2 as Novel Therapeutic Target in Fibrotic Diseases.

G protein-coupled receptor kinase 2 (GRK2), an important subtype of GRKs, specifically phosphorylates agonist-activated G protein-coupled receptors (GPCRs). Besides, current research confirms that it participates in multiple regulation of diverse cells via a non-phosphorylated pathway, including interacting with various non-receptor substrates and binding partners. Fibrosis is a common pathophysiological phenomenon in the repair process of many tissues due to various pathogenic factors such as inflammation, injury, drugs, etc. The characteristics of fibrosis are the activation of fibroblasts leading to myofibroblast proliferation and differentiation, subsequent aggerate excessive deposition of extracellular matrix (ECM). Then, a positive feedback loop is occurred between tissue stiffness caused by ECM and fibroblasts, ultimately resulting in distortion of organ architecture and function. At present, GRK2, which has been described as a multifunctional protein, regulates copious signaling pathways under pathophysiological conditions correlated with fibrotic diseases. Along with GRK2-mediated regulation, there are diverse effects on the growth and apoptosis of different cells, inflammatory response and deposition of ECM, which are essential in organ fibrosis progression. This review is to highlight the relationship between GRK2 and fibrotic diseases based on recent research. It is becoming more convincing that GRK2 could be considered as a potential therapeutic target in many fibrotic diseases.

ß-arrestin2 regulating ß2-adrenergic receptor signaling in hepatic stellate cells contributes to hepatocellular carcinoma progression.

Background: ß-arrestin2 and ß2-adrenergic receptor (ß2-AR) have important roles in malignant tumors, the present study aims to investigate the role of activated ß2-AR in hepatic stellate cells (HSCs) during hepatocellular carcinoma (HCC) progression and the regulatory effect of ß-arrestin2. Methods: Immunofluorescence and Western blot were used to detect the expression of ß-arrestin2 and ß2-AR in HSCs of liver tissues from human HCC samples and diethylnitrosamine (DEN)-induced HCC model mice. We next used ß-arrestin2-/- mice to demonstrate the regulatory role of ß-arrestin2 in DEN mice. The subsets of T cells were quantified by flow cytometry. MTT and wound healing assay were applied to detect the proliferation and migration of cells. Co-immunoprecipitation assay was used to detect the link of ß-arrestin2 and ß2-AR in HSCs. Effect of ß-arrestin2 overexpression on ß2-AR downstream signaling pathway was verified by Western blot. The secretion of CCL2 was detected by ELISA. Results: The expression of ß2-AR was significantly increased, while ß-arrestin2 was decreased in HSCs of HCC tissues. And ß-arrestin2 deficiency exacerbates DEN-induced HCC accompanied with increased ß2-AR expression. The results of flow cytometry showed that the percentage of activated T cells decreased gradually after DEN injection. ß-arrestin2 knockout down-regulated the ratio of activated T cells. In vitro, selective activation of ß2-AR in HSCs promoted the proliferation and migration of HCC cells. ß-arrestin2 overexpression enhanced co-immunoprecipitation of ß-arrestin2 and ß2-AR in activated HSCs, and decreased its downstream Akt phosphorylation. Akt inhibitor decreased secretion of CCL2 in activated HSCs. Conclusion: Our study demonstrated that ß2-AR activation in HSCs induces the proliferation and migration of HCC cells may be through Akt signaling, and this effect appears to be regulated by ß-arrestin2.

ß-Arrestin2 deficiency attenuates oxidative stress in mouse hepatic fibrosis through modulation of NOX4.

Hepatic fibrosis is a disease characterized by excessive deposition of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is responsible for most of ECM production. Oxidative stress and reactive oxygen species (ROS) may be important factors leading to liver fibrosis. NADPH oxidase 4 (NOX4) is the main source of ROS in hepatic fibrosis, but the mechanism by which NOX4 regulates oxidative stress is not fully understood. ß-Arrestin2 is a multifunctional scaffold protein that regulates receptor endocytosis, signaling and trafficking. In this study, we investigated whether ß-arrestin2 regulated oxidative stress in hepatic fibrosis. Both ß-arrestin2 knockout (Arrb2 KO) mice and wild-type mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce hepatic fibrosis. Arrb2 KO mice showed significantly attenuated liver fibrosis, decreased ROS levels and NOX4 expression, and reduced collagen levels in their livers. In vitro, NOX4 knockdown significantly inhibited ROS production, and decreased expression of alpha-smooth muscle actin in angiotensin II-stimulated human HSC cell line LX-2. Through overexpression or depletion of ß-arrestin2 in LX-2 cells, we revealed that decreased ß-arrestin2 inhibited ROS levels and NOX4 expression, and reduced collagen production; it also inhibited activation of ERK and JNK signaling pathways. These results demonstrate that ß-arrestin2 deficiency protects against liver fibrosis by downregulating ROS production through NOX4. This effect appears to be mediated by ERK and JNK signaling pathways. Thus, targeted inhibition of ß-arrestin2 might reduce oxidative stress and inhibit the progression of liver fibrosis.

To explore the therapeutic effect of Kangfuxiaomi shuan II on cervicitis in rats

ABSTRACT Purpose: The rat cervicitis model was established with 20% phenol glue to explore the therapeutic effect of Kangfuxiaomi shuan II on rat cervicitis and its mechanism. Methods: After modeling, the rats were treated with Shuangzuotai suppository (37.84 mg/kg), Kangfuxiaoyan shuan (205.6 mg/kg) and Kangfuxiaomi shuan II (40, 80, 160 mg/kg). The histopathological changes and injury degree of cervix in rats were evaluated by vulvar inflammation score and organ index. The therapeutic effect of Kangfuxiaomi shuan II on cervicitis was evaluated by detecting the levels of copper-protein (CP), C-reactive protein (CRP), Rat interleukin 6 (IL-6), superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and epidermal growth factor (EGF), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cervical tissue. Results: Compared with the model group, the vulvar inflammation score and cervical index of rats in other groups decreased significantly (P<0.01). Kangfuxiaomi shuan II could significantly reduce the levels of CP, CRP, and MDA in serum of rats with cervicitis, and significantly increase the activity of SOD in serum of rats with cervicitis (P<0.01). The levels of EGF and iNOS in cervical tissue of rats also increased in different degrees, while the level of COX-2 decreased significantly (P<0.01), which significantly improved the pathological degree of vulvar inflammation in rats with cervicitis. Conclusions: Kangfuxiaomi shuan II has a certain therapeutic effect on cervicitis in rats, and its mechanism may be related to the regulation of inflammatory cytokine network and immunity.

GRK2 Suppresses Hepatocellular Carcinoma Metastasis and Invasion Through Down-Regulation of Prostaglandin E Receptor 2.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive form of human liver cancer and the fifth most common malignancy worldwide. Novel effective treatment strategies for HCC are urgently in clinical because of its poor response to conventional therapies. G protein-coupled receptor kinases (GRKs), including GRK2 and GRK3, are known that involves in various essential cellular processes and regulates numerous signaling pathways. However, the role of GRK2/3 in invasion and metastasis of HCC still remains unclear. MATERIALS AND METHODS: Immunohistochemistry, Western blot, laser confocal microscopy and qRT-PCR were used to detect the expression of GRK2/3 and EP2 in liver tissues of HCC patients and DEN-induced HCC mice. Wound healing and transwell assay were applied to measure the migration and invasion of HCC cells after transfected with GRK2 siRNA. The downstream pathway of Akt and ERK was verified by Western blot. RESULTS: The expression of GRK2 was significantly decreased, while GRK3 was not significantly changed in HCC tissues compared with noncancerous tissues of HCC patients. Moreover, GRK2 expression was reduced during liver tumorigenesis in diethylnitrosamine-induced liver tumor model. In addition, our in vitro study showed that GRK2 expression was gradually decreased with increasing HCC cell line metastatic potential, and GRK2 knockdown significantly promoted the migration and invasion of HCC cells. Furthermore, low GRK2 expression was associated with increased expression of EP2 receptor translocation to HCC cell membrane, and the activation of Akt pathway. CONCLUSION: These data suggest that GRK2 inhibits HCC metastasis and invasion may be through regulating EP2 receptor translocation, and this effect appears to be mediated by Akt pathway.