Study on therapeutic mechanism of total salvianolic acids against myocardial ischemia-reperfusion injury based on network pharmacology, molecular docking, and experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Salviae miltiorrhizae, also known as Danshen in Chinese, effectively activates the blood and resolves stasis. Total salvianolic acids (SA) is the main active ingredient of Danshen, and related preparations, such as salvianolate injection are commonly used clinically to treat myocardial ischemia-reperfusion injury (MIRI). However, the potential targets and key active ingredients of SA have not been sufficiently investigated. AIM OF THE STUDY: This study aimed to investigate the mechanism of action of SA in treating MIRI. MATERIALS AND METHODS: Network pharmacology and molecular docking techniques were used to predict SA targets against MIRI. The key acting pathway of SA were validated by performing experiments in a rat MIRI model. RESULTS: Twenty potential ingredients and 54 targets of SA in treating MIRI were identified. Ingredient-target-pathway network analysis revealed that salvianolic acid B and rosmarinic acid had the highest degree value. Pathway enrichment analysis showed that SA may regulate MIRI through the IL-17 signaling pathway, and this result was confirmed in the rat MIRI experiment. CONCLUSION: The results of this study indicate that SA may protect MIRI by regulating the IL-17 pathway.

[Research progress on the biological effects of HIF-1α on follicle development and ovulation].

Hypoxia inducible factor-1α (HIF-1α), as a hypoxia inducible factor, affects women's reproductive function by regulating the development and excretion of follicles. HIF-1α induces glycolysis and autophagy in the granule cells by promoting oocyte development, regulating the secretion of related angiogenic factors, and improving follicle maturity. In addition, HIF-1α promotes the process of luteinization of follicular vesicles, maintains luteal function, and finally completes physiological luteal atrophy through cumulative oxidative stress. Dysfunction of HIF-1α will cause a series of pathological consequences, such as angiogenesis defect, energy metabolism abnormality, excessive oxidative stress and dysregulated autophagy and apoptosis, resulting in ovulation problem and infertility. This article summarizes the previous studies on the regulation of follicle development and excretion and maintenance of luteal function and structural atrophy by HIF-1α. We also describe the effective intervention mechanism of related drugs or bioactive ingredients on follicular dysplasia and ovulation disorders through HIF-1α, in order to provide a systematic and in-depth insights for solving ovulation disorder infertility.

[Mechanism of tonifying kidney and activating blood therapy for premature ovarian failure:a review].

Healthy birth and child development are the prerequisite for improving the overall quality of the population. However, premature ovarian failure(POF) threatens the reproductive health of women. The incidence of this disease has been on the rise, and it tends to occur in the young. The causes are complex, involving genetics, autoimmune, infectious and iatrogenic factors, but most of the causes remain unclear. At the moment, hormone replacement therapy and assisted reproductive technology are the main clinical approaches. According to traditional Chinese medicine(TCM), kidney deficiency and blood stasis are one of the major causes of POF, and TCM with the effects of tonifying kidney and activating blood has a definite effect. Through clinical trials, TCM prescriptions for POF have excellent therapeutic effect as a result of multi-target regulation and slight toxicity. In particular, they have no obvious side effects. A large number of studies have shown that the kidney-tonifying and blood-activating TCM can regulate the neuroendocrine function of hypothalamic-pituitary-ovarian axis, improve ovarian hemodynamics and microcirculation, reduce the apoptosis of granulosa cells, alleviate oxidative stress injury, and modulate immunologic balance. The mechanism is that it regulates the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt), vascular endothelial growth factor(VEGF), transforming growth factor(TGF)-ß/Smads, nuclear factor E2-related factor 2(Nrf2)/antioxidant response element(ARE), and nuclear factor-kappa B(NF-κB) signaling pathways. This article summarized the pathological mechanisms of tonifying kidney and activating blood TCM in the prevention and treatment of POF and explored the biological basis of its multi-pathway and multi-target characteristics in the treatment of this disease. As a result, this study is expected to serve as a reference for the treatment of POF with the tonifying kidney and activating blood therapy.

Chinese Herbal Preparation SaiLuoTong Alleviates Brain Ischemia via Nrf2 Antioxidation Pathway-Dependent Cerebral Microvascular Protection.

Stroke is one of the most devastating diseases worldwide. The Chinese herbal preparation SaiLuoTong (SLT) capsule showed outstanding therapeutic effects on stroke and its sequelae. The aim of this study was to further elucidate its therapeutic mechanism. We duplicated a permanent cerebral ischemia model in rats by MCAO and used SLT (33 and 16.5 mg/kg) to intervene. The results showed SLT dose dependently decreased infarction volumes, relieved neuron degeneration and loss, and ameliorated neurological functions, and the dose of 33 mg/kg had statistical significance (compared with the model group, p < 0.05); SLT of 33 mg/kg also significantly inhibited the elevation in brain water content and the loss in claudin-1 and occludin expressions; additionally, it significantly increased nucleus translocation of Nrf2, elevated the expression of HO-1, and raised the activity of SOD and content of GSH (compared with the model group, p < 0.05 or 0.01). These results testified SLT's anti-brain ischemia effect and hint this effect may be related to the protection of brain microvascular endothelial cells (BMECs) that is dependent on the Nrf2 pathway. To further testify, we cultured hCMEC/D3 cells, duplicated OGD/R model to simulate ischemia, and used SLT (3.125, 6.25, and 12.5 mg/L) to treat. SLT dose dependently and significantly inhibited the drop in cell viabilities, and activated the Nrf2 pathway by facilitating Nrf2 nucleus translocation, and increasing HO-1 expression, SOD activity, and GSH content (compared with the model group, p < 0.05 or 0.01); last, the anti-OGD/R effects of SLT, including raising cell viabilities, inhibiting the elevation in dextran permeability, and preserving expressions of claudin-1 and occludin, were all abolished by Nrf2 siRNA interference. The in vitro experiment undoubtedly confirmed the direct protective effect of SLT on BMECs and the obligatory role of the Nrf2 pathway in it. Collectively, data of this study suggest that SLT's therapeutic effect on brain ischemia is related to its Nrf2-dependent BMECs protection.

Enterovirus D68 in hospitalized children with respiratory symptoms in Guangdong from 2014 to 2018: Molecular epidemiology and clinical characteristics.

BACKGROUND: Enterovirus D68 (EV-D68) is an emerging pathogen in humans. EV-D68 causes a wide range of respiratory symptoms in children and has the propensity to cause severe complications. EV-D68 outbreaks are rarely investigated in mainland China. Therefore, in this study, we aimed to investigate the prevalence of EV-D68 in children and to describe the clinical manifestations as well as the phylogeny of EV-D68 in Guangdong Province from 2014 to 2018. METHODS: Nasopharyngeal swabs were collected from hospitalized children with respiratory symptoms and screened for respiratory pathogens by fluorescence quantitative PCR and culture. The EV-positive samples were subsequently typed by sequencing the 5'-untranslated region and EV-D68-specific VP1 capsid gene. A phylogenetic tree was constructed by the maximum-likelihood method based on the VP1 gene using ClustalW. RESULTS: A total of 1,498 (59.8%) out of 2,503 children were screened positive for ≥1 virus species. Among the 158 (6.31%) EV-positive samples, 17 (0.68%) were identified as EV-D68. Most EV-D68 cases (n = 14) were diagnosed with pneumonia and bronchial pneumonia. No deaths were found in EV-D68 cases. Wheezing occurred in EV-D68 cases more frequently (70.59% vs. 43.26%, P = 0.040) than that of other EVs. All the EV-D68 were of clade B3, which were highly similar to the strains circulating in China. CONCLUSION: EV-D68 was the predominant enterovirus type in hospitalized children with respiratory symptoms in Guangdong Province. All the EV-D68 strains belong to clade B3. The development of diagnostic tools is warranted in order to monitor EV-D68 infections in China.

Berberine Protects against TNF-α-Induced Injury of Human Umbilical Vein Endothelial Cells via the AMPK/NF-κB/YY1 Signaling Pathway.

Endothelial injury, characterized by an inflammatory response and increased permeability, is an initial stage of atherosclerosis (AS). Adenosine 5'-monophosphate (AMP), activated protein kinase (AMPK), and Nuclear Factor kappa B (NF-κB)/Yin Yang 1(YY1) signaling pathways play important roles in the process of endothelial injury. Berberine (BBR), a bioactive alkaloid isolated from several herbal substances, possesses multiple pharmacological effects, including anti-inflammatory, antimicrobial, antidiabetic, anticancer, and antioxidant activities. Previous studies showed a protective effect of berberine against endothelial injury. However, the underlying mechanism remains unclear. We explored the potential effect of BBR on TNF- (tumor necrosis factor-) α-induced injury of human umbilical endothelial cells (HUVECs) and studied its possible molecular mechanism. In the present study, HUVECs were divided into three groups. HUVEC viability was measured with Cell Counting Kit-8 assay. Extracellular lactic dehydrogenase (LDH) concentration was measured with LDH leakage assay. Endothelial microparticle (EMP) numbers were evaluated by flow cytometry analysis assay. The expression of proinflammatory cytokines was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). The mRNA expression of NF-κB and YY1 was detected by Real-Time PCR (RT-PCR). The protein expression of NF-κB, YY1, and AMPK was detected by immunofluorescence microscopy assay or western blot analysis. The results showed that LDH concentration, EMPs numbers, and the expression of proinflammatory cytokines (IL-6, IL-8, and IL-1ß) increased in TNF-α-induced injured HUVECs, but ameliorated by BBR pretreatment. BBR pretreatment upregulated the expression of phosphorylated AMPK and downregulated the expressions of NF-κB and YY1 in injured HUVECs induced by TNF-α, which were offset by the AMPK inhibitor Compound C (CC). The results indicated that BBR protected against TNF-α-induced endothelial injury via the AMPK/NF-κB/YY1 signaling pathway.

HDAC9 promotes brain ischemic injury by provoking IκBα/NF-κB and MAPKs signaling pathways.

Ischemic stroke is an acute cerebrovascular disease due to poor blood flow to the brain. Nevertheless, there is still no effective therapy for it and the pathology contributing to ischemic stroke is not fully understood. Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme. HDAC9 gene region is a leading risk locus for large artery atherosclerotic stroke. However, the mechanisms linking HDAC9 to ischemic remain elusive. In the study, we attempted to explore HDAC9-associated inflammatory response using the wild type (WT) and HDAC9-knockout (KO) mice with brain ischemic injury. The results indicated that WT mice with ischemia brain exhibited higher expression levels of HDAC9. HDAC9 depletion resulted in a decreased infarct volume and an improved neurological function in mice after ischemic reperfusion (I/R) injury. I/R injury markedly enhanced GFAP and Iba-1 expressions in cortex and HDAC9 knockout significantly reversed this up-regulation. Loss of HDAC9 inhibited the release of inducible NO-synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), and IL-18 in cortex, hippocampus and hypothalamus of mice with I/R injury, which occurred at the transcription levels. Furthermore, the inhibitory actions of HDAC9 deficiency were associated with the down-regulation of phosphorylated-IκBα, phosphorylated-nuclear factor-kappa B (NF-κB), and p-mitogen-activated protein kinases (MAPKs), including phosphorylated-p38, phosphorylated-extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated-c-Jun N-terminal kinase (JNK). Importantly, the in vitro study indicated that HDAC9 inhibition-reduced inflammation and activation of IκBα/NF-κB were restored by promoting MAPKs activity in LPS-stimulated cells. Our findings suggest that HDAC9 inhibition showed neuroprotective effects on ischemic stroke by restraining inflammation, which might help develop new and effective strategies for the therapeutic interventions in ischemic stroke.

Increase of SOX9 promotes hepatic ischemia/reperfusion (IR) injury by activating TGF-ß1.

Ischemia/reperfusion (IR) injury causes damage in aerobically metabolizing organs or tissues, which is an essential injury mechanism in various clinical settings. SRY-related high mobility group-Box gene 9 (SOX9) is a transcription factor of the SRY family, modulating various cellular processes, including fibrosis formation and tumor growth. However, the effects of SOX9 on hepatic IR injury have not been explored. In the present study, a hepatic IR injury model was established, supported by a significant histological alteration with high Suzuki scores, and a remarkable up-regulation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Importantly, we found that SOX9 was over-expressed in liver of mice after IR operation. Suppressing SOX9 markedly reduced inflammatory response, as evidenced by the reduced mRNA expressions of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1ß and inactivation of inhibitor of κBα (IκBα)/nuclear factor (NF)-κB pathway. In addition, SOX9 suppression alleviated apoptosis in liver of mice after IR injury, as supported by the reduced number of terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL)-staining cells and decreased expression of Caspase-3 in liver tissue sections. The role of SOX9 in accelerating hepatic IR injury was further confirmed in primary hepatocytes under hypoxiaand reoxygenation (HR) treatment by enhancing inflammatory response and apoptosis. Of note, we found that transforming growth factor (TGF)-ß1 was highly induced in liver of mice after IR injury. HR treatment also stimulated TGF-ß1 expressions in vitro. Significantly, SOX9 over-expression-induced inflammation and apoptosis were obviously reduced by pirfenidone (Pirf), TGF-ß1 inhibitor. In contrast, TGF-ß1 exposure to cells further enhanced inflammation and apoptosis in HR-operated cells either with SOX9 knockdown or over-expression. Therefore, we identified a novel SOX9-dependent pathway that contributed to hepatic IR injury through enhancing inflammation and apoptosis by activating TGF-ß1.

Suppression of Elp2 prevents renal fibrosis and inflammation induced by unilateral ureter obstruction (UUO) via inactivating Stat3-regulated TGF-ß1 and NF-κB pathways.

Renal fibrosis and inflammation are common underlying processes of progressive kidney diseases. Elongator protein 2 (Elp2), identical to signal transducer and activator of transcription-3 (STAT-3)-interacting protein-1 (Stip1), is a component of the Elongator complex that regulates RNA polymerase II. Elp2 regulates STAT-3 activation to control various cellular processes. The mechanisms of Elp2 prevention in renal interstitial fibrosis and inflammation remain unknown. In the study, Elp2 transgenic knockout (KO) and wild type (WT) mice were employed to investigate the effects of Elp2 on renal fibrosis and inflammation development after unilateral ureter obstruction (UUO) surgery. The results indicted that Elp2 was significantly expressed in renal tissues of WT/UUO mice. Elp2-KO mice exhibited attenuated histological changes of kidney, as well as collagen and fibrosis accumulation. Lower expressions of transforming growth factor (TGF)-ß1, α-smooth muscle actin (α-SMA), fibronectin, vimentin, and phospho-Smad2/3 were observed in kidney of Elp2-KO mice than that of WT mice after UUO. Elp2-KO mice showed less inflammation, as evidenced by the decrease of circulating or renal pro-inflammatory cytokines, as well as the reduction of phospho-nuclear factor (NF)-κB. Additionally, Elp2-KO apparently led to a decrease in phospho-STAT3 in kidney of UUO mice. In vitro, we found that TGF-ß1- and LPS-induced fibrosis and inflammation were abrogated by Elp2 knockdown, which were intriguingly abolished by activating STAT3 phosphorylation using its activator of colivelin (Col). Together, our findings supplied that Elp2 might be a potential therapeutic target to prevent the progression of renal fibrosis and inflammation.

HDAC11 deletion reduces fructose-induced cardiac dyslipidemia, apoptosis and inflammation by attenuating oxidative stress injury.

Diabetes mellitus (DM) is a risk factor for abnormal heart development, but the molecular mechanism remains obscure. Histone deacetylase 11 (HDAC11), the most recently identified histone deacetylase, is the sole member of class IV HDACs. However, its role in diabetic cardiac injury is still poorly understood. In the present study, we attempted to explore the effects of HDAC11 on fructose (Fru)-induced cardiac injury using the wild type (HDAC11+/+) and knockout (HDAC11-/-) mice. The results indicated that HDAC11 was significantly expressed in human and mouse diabetic heart failure (DHF) hearts. HDAC11-/- reduced the body weight, inguinal fat-pad mass, and elevated blood pressure in Fru-fed mice. Compared to HDAC11+/+/Fru group, cardiac function was significantly improved in HDAC11-/-/Fru mice. HDAC11-/-/Fru mice exhibited reduced cardiac triacylglycerol (TG), total cholesterol (TC) and free fatty acid (FFA) levels, along with decreased mRNA levels of lipid synthesis-, lipid storage- and lipid oxidation-associated genes. In addition, HDAC11-/- attenuated apoptosis, oxidative stress and inflammation in the heart of Fru-fed mice, as evidenced by the reduced cleavage of Caspase-3, nicotinamide adenine dinucleotide phosphate (NADPH), and xanthine oxidase (XOD) activity, enhanced superoxide dismutase (SOD) activity, as well as the decreased interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) levels, which was accompanied with down-regulated p-NF-κB. The results above were verified in Fru-treated primary cardiomyocytes isolated from HDAC11+/+ or HDAC11-/- mice. Intriguingly, suppressing the expressions of anti-oxidants using zinc protoporphyrin (ZnPP) or siNrf-2 siRNA markedly abolished the results that HDAC11 suppression-induced reduction of apoptosis, reactive oxygen species (ROS) production, inflammation, as well as the improvement of dyslipidemia in Fru-incubated primary cardiomyocytes. Thus, ROS production was responsible for HDAC11-modulated diabetic heart injury. These findings suggested that suppressing HDAC11 has therapeutic potential for treating diabetes mellitus-associated cardiac injury.

[Observation on clinical efficacy of activating renal blood circulation and ovarian stimulation formula in treating ovulation failure infertility].

OBJECTIVE: To discuss the clinical efficacy of the activating renal blood circulation and ovarian stimulation formula in treating ovulation failure infertility. METHOD: Eighty-six cases were randomly divided into two groups: the treatment group and the control group. The treatment group is administered with the activating renal blood circulation and ovarian stimulation formula (composed of 15 g Cuscutae Semen, 15 g Dipsaci Asperoidis Radix, 15 g Lycii Fructus, 15 g Spatholobi Caulis, 10 g Ligustri Lucidi Fructus, 15 g Lycopi Herba, 10 g Typhae Polleu, 10 g Angelicae Sinensis Radix, 15 g Cyathulae Radix etc.), whereas the control group was given clomiphene. RESULT: The treatment group showed a pregnancy rate of 58.14%, with an ovulation rate of 68.6%. While the control group showed a pregnancy rate of 36.67%, with an ovulation rate of 70%. CONCLUSION: The comparison between the two groups showed that the activating renal blood circulation and ovarian stimulation formula was significantly different from clomiphene in statistical analysis (P < 0.05), without notable difference in the ovulation rate. Before and after the treatment, there is no significant difference in diameter of dominant follicles between the two groups, with remarkable difference in endometrium (P < 0.05).

[Systematic evaluation of kidney-tonifying and blood-activating traditional Chinese medicines in treatment of ovulatory disorder infertility].

To evaluate clinical studies on effect of traditional Chinese medicines in treating ovulatory disorder infertility by using evidence-based medical method. According to the standard of Cochrane Handbook, the selection standard of the randomized controlled clinical trail on kidney-tonifying and blood-activating traditional Chinese medicines in the treatment of ovulatory disorder infertility was formulated. Literatures in line with the standard were searched through computers among Chinese and English databases. The literatures included were evaluated by evidence-based medicine method, and analyzed with Revman 5. 1. There were 19 articles in line with the standard. Data for pregnancy rate, ovulation rate and miscarriage rate was extracted from them, and heterogeneity test was conducted with Meta, which showed no statistical heterogeneity. Results showed pregnancy rate (n = 17) , RRp 1. 66, 95% C1 [1. 45, 1.92], Z7.06 (P<0.000 01); ovulation rate (n =11) , RRo 1.05, 95% CI[0.97,1. 12], Z 1.25 (P=0.21); miscarriage rate (n =5) , RRM, 0. 24, 95% CI[0. 11,0. 56] , Z 3.33 (P =0. 000 9). After analysis and comparison, it is found that the Chinese medical treatment group was superior to the Western medicine control group in terms of pregnancy rate, which showed statistic significance. Both groups showed no obvious difference in the ovulation rate, and no statistic significance; the miscarriage rate of the Chinese medicine group is obviously lower than that of the Western medicine control group, which indicated statistic significances. This study preliminarily proves the good efficacy and unique advantages of kidney-tonifying and blood-activating traditional Chinese medicines in treating ovulatory disorder infertility.