A novel quantification method of lung fibrosis based on Micro-CT images developed with the optimized pulmonary fibrosis mice model induced by bleomycin.

Background and aims: Idiopathic pulmonary fibrosis (IPF) is a fibrosing lung disease with unknown etiology, leading to cough and dyspnoea, which is also one of the most common sequelae affecting the quality of life of COVID-19 survivors. There is no cure for IPF patients. We aim to develop a reliable IPF animal model with quantification of fibrosis based on Micro-Computer Tomography (micro-CT) images for the new drug discovery, because different bleomycin administration routes, doses, and intervals are reported in the literature, and there is no quantitative assessment of pulmonary fibrosis based on micro-CT images in animal studies. Methods: We compared three dosages (1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg) of intratracheal bleomycin administration and experiment intervals (14 and 21 days) in C57BL/6 mice by investigating survival rates, pulmonary histopathology, micro-CT, peripheral CD4+ & CD8+ cells, and cytokines. Moreover, a simple and reliable new method was developed for scoring fibrosis in live mice based on Micro-CT images by using Image J software, which transfers the dark sections in pulmonary Micro-CT images to light colors on a black background. Results: The levels of hydroxyproline, inflammation cytokine, fibrotic pathological changes, and collagen deposition in the lungs of mice were bleomycin dose-dependent and time-dependent as well as the body weight loss. Based on the above results, the mice model at 21 days after being given bleomycin at 1.25 mg/kg has optimal pulmonary fibrosis with a high survival rate and low toxicity. There is a significant decrease in the light area (gray value at 9.86 ± 0.72) in the BLM mice, indicating that a significant decrease in the alveolar air area was observed in BLM injured mice compared to normal groups (###p < 0.001), while the Pirfenidone administration increased the light area (gray value) to 21.71 ± 2.95 which is close to the value observed in the normal mice (gray value at 23.23 ± 1.66), which is consistent with the protein levels of Col1A1, and α-SMA. Notably, the standard deviations for the consecutive six images of each group indicate the precision of this developed quantitation method for the micro-CT image taken at the fifth rib of each mouse. Conclusion: Provided a quantifying method for Micro-CT images in an optimal and repeatable pulmonary fibrosis mice model for exploring novel therapeutic interventions.

Circular RNA circRNA_101996 promoted cervical cancer development by regulating miR-1236-3p/TRIM37 axis.

Circular RNAs (circRNAs) appear to be significant modulators in various physiological processes. Recently, it is found that circRNA_101996 exerts important roles in various cancers. Our previous studies showed that circRNA_101996 promoted cervical cancer growth and metastasis by regulating miR-8075/TPX2. However, the potential regulatory role of circRNA_101996 in cervical cancer still needs further investigation. Our results in this study suggested that circRNA_101996 was over-expressed in cervical cancer patients. circRNA_101996 up-regulation remarkably assisted cell proliferation, cell cycle progression, and cell migration in cervical cancer, while circRNA_101996 knockdown exerted the inverse effects. The molecular investigations indicated that circRNA_101996 could increase the expression level of miR-1236-3p, tripartite motif-containing 37 (TRIM37), through binding to miR-1236-3p and reducing its expression. Moreover, in vivo results demonstrated that circRNA_101996 shRNA can function as a tumor suppressor through down-regulating TRIM37 in cervical cancer. In conclusion, our data indicated that circRNA_101996/miR-1236-3p/TRIM37 axis accelerated cervical cancer development, providing novel insights into cervical cancer diagnosis and treatment.

Corrigendum to "Selection of quality markers of Jasminum amplexicaule based on its anti-diarrheal and anti-inflammatory activities: Effect-target affiliation-traceability-pharmacokinetics strategy" [Chinese Herbal Medicines 11 (2019) 379-386].

[This corrects the article DOI: 10.1016/j.chmed.2019.08.002.].

Pro-inflammatory cytokines in the paraventricular nucleus mediate the adipose afferent reflex in rats.

Our previous study showed that the adipose afferent reflex (AAR) induced by chemical stimulation of white adipose tissue (WAT) increased sympathetic outflow and blood pressure. We also found that pro-inflammatory cytokines (PICs) in the hypothalamic paraventricular nucleus (PVN) potentiate the cardiac sympathetic afferent reflex in rats. However, the role of PICs in the PVN in regulating the AAR is still not clear. This study determined whether PICs in the PVN mediate the AAR in rats. The AAR was evaluated based on renal sympathetic nerve activity and mean arterial blood pressure in response to capsaicin injection into inguinal WAT (iWAT). PIC levels were measured by ELISA. PVN microinjection with the PICs tumor necrosis factor (TNF)-α or interleukin (IL)-1ß enhanced the AAR in a dose-dependent manner. Furthermore, pretreatment via the bilateral microinjection of the TNF-α-blocker etanercept or IL-1ß blocker IL-1ra into the PVN attenuated the AAR. In rats pretreated with TNF-α or IL-1ß, a sub-response dose of angiotensin II (Ang II) significantly enhanced the AAR. Moreover, delivery of the angiotensin II type 1(AT1) receptor antagonist losartan into the PVN attenuated the effects of TNF-α or IL-1ß on the AAR. In addition, stimulating either iWAT or retroperitoneal WAT with capsaicin increased TNF-α or IL-1ß levels in the PVN, but the injection of capsaicin into the jugular vein, skeletal muscle, and skin had no effects on TNF-α or IL-1ß levels in the PVN. These results suggest that TNF-α or IL-1ß and Ang II in the PVN synergistically enhance the AAR in rats.

[The protective effects of lycium barbarum polysaccharides on retinal neurons in diabetic rats and its mechanism].

OBJECTIVE: To clarify whether lycium barbarum polysaccharides (LBP) have protective effects on retina neuronal cells in diabetic rats and to identify the related mechanism involved in this process. METHODS: Eighteen SD rats were randomly divided into 3 groups ( n= 6): normal control group (NC), diabetes mellitus group (DM) and LBP-treatment group (DM+LBP). The diabetic rat model was induced by single intraperitoneal injection of streptozotocin (STZ). The rats in DM+LBP group were treated with LBP at the dose of 1 mg/kg by gavage, once a day for 12 weeks. After the treatment, the weight and blood glucose, the generation of reactive oxygen species (ROS), the surviving retinal ganglion cells (RGCs) and amacrine cells and the protein expressions of nuclear factor E2-related factor 2 (Nrf2) and the heme oxygenase-1 (HO-1) were detected. RESULTS: The successful rate of diabetic model was 100%. Compared with NC group, the rats of DM group caused weight loss, elevated blood glucose, a marked increase of ROS generation and a significant decrease in the number of RGCs and amacrine cells (P＜0.01), and these effects were diminished or abolished by LBP treatment. Meanwhile, LBP significantly increased the expressions of Nrf2 and HO-1 in the retinas of diabetic rats (P＜0.01). CONCLUSION: LBP can improve retinal oxidative stress and exert beneficial neuroprotective effects in diabetic rats, and its mechanism may be associated with the activation of the Nrf2/HO-1 antioxidant pathway.

Short hairpin RNA interference targeting interleukin 1 receptor type I in the paraventricular nucleus attenuates hypertension in rats.

Blood pressure is controlled by tonic sympathetic activities, excessive activation of which contributes to the pathogenesis and progression of hypertension. Interleukin (IL)-1ß in the paraventricular nucleus (PVN) is involved in sympathetic overdrive and hypertension. Here, we investigated the therapeutic effects of IL-1 receptor type I (IL-1R1) gene silencing in the PVN on hypertension. Recombinant lentivirus vectors expressing a short hairpin RNA (shRNA) targeting IL-1R1 (Lv-shR-IL-1R1) or a control shRNA were microinjected into PVN of spontaneously hypertensive rats (SHRs) and normotensive WKY rats. The fluorescence of green fluorescent protein-labelled vectors appeared at 2 weeks after injection and persisted for at least 8 weeks. IL-1R1 protein expression in the PVN was reduced 4 weeks after Lv-shR-IL-1R1 injection in SHRs. IL-1R1 interference also reduced basal sympathetic activity, cardiac sympathetic afferent reflex in SHRs. Depressor effects were observed from week 2 to 10 after Lv-shR-IL-1R1 treatment in SHRs, with the most prominent effects seen at the end of week 4. Furthermore, Lv-shR-IL-1R1 treatment decreased the ratio of left ventricular weight to body weight and cross-sectional areas of myocardial cells in SHRs. Additionally, Lv-shR-IL-1R1 treatment prevented an increase in superoxide anion and pro-inflammatory cytokines (PICs, TNF-α and IL-1ß) in the PVN of SHR, and upregulated anti-inflammatory cytokine (AIC, IL-10) expression. These results indicate that shRNA interference targeting IL-1R1 in the PVN decreases arterial blood pressure, attenuates excessive sympathetic activity and cardiac sympathetic afferent reflex, and improves myocardial remodelling in SHRs by restoring the balance between PICs and AICs to attenuate oxidative stress.

Neuroprotective effects of total flavonoid fraction of the Epimedium koreanum Nakai extract on dopaminergic neurons: In vivo and in vitro.

Flavonoids, the active components of Epimedii Genus, have been demonstrated to protect against osteoporosis, cardiovascular diseases and rheumatoid arthritis. The present study aimed to investigate the neuroprotective effects of total flavonoid (TF) fraction of Epimedium koreanum Nakai on dopaminergic neurons in the cellular and mice models of Parkinson's disease (PD). TF pretreatment could ameliorate the decrease of striatal dopamine (DA) content and the loss of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra pars compacta (SNpc) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). TF treatment could reverse the changes of Bcl-2 and Bax protein expressions in the striatum of PD mice. 1-Methyl-4-phenylpyridinium ion (MPP+) significantly decreased the cell viability and mitochondrial membrane potential in MES23.5 cells. These effects could be reversed by TF treatment. In addition, MPP+-induced changes of Bcl-2 and Bax mRNA and protein expressions were also reversed by TF pretreatment. These data demonstrated that TF of E. koreanum Nakai could protect against MPTP-induced dopaminergic neuronal death in mice and MPP+-induced neurotoxicity in dopaminergic MES23.5 cells. Anti-apoptosis might be involved in this process.

Neuroprotective properties of icariin in MPTP-induced mouse model of Parkinson's disease: Involvement of PI3K/Akt and MEK/ERK signaling pathways.

BACKGROUND: Epimedium sagittatum is a traditional Chinese herb normally which is used to treat the osteoporosis, cardiovascular dysfunction, and to improve neurological and sexual function in China, Korea and Japan. Icariin is the major active ingredient in Epimedium sagittatum. In the present research, we examined the neuroprotective effects of icariin on dopaminergic neurons and the possible mechanisms in a mouse model of Parkinson's disease (PD). METHODS: Ovariectomized PD mice were treated with vehicle or icariin (3 days before MPTP injections) with or without the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or mitogen-activated protein kinase kinase (MEK) inhibitor PD98059. The dopamine (DA) content in the striatum was studied by HPLC. Western blot was used to determine the protein expressions of Bcl-2, Bax and Caspase 3 in the striatum. The numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantial nigra pars compacta (SNpc) were assessed by immunohistochemistry. The activation of Akt and ERK by icariin were detected in doparminergic MES23.5 cells. RESULTS: Icariin pretreatment could ameliorate the decreased striatum DA content and the loss of TH-IR neurons in the SNpc induced by MPTP. The MPTP-induced changes of Bcl-2, Bax and caspase 3 protein expressions in the striatum could be reversed by icariin pretreatment. Blockade of PI3K/Akt or MEK/ERK signaling pathway by LY294002 or PD98059 could attenuate the increase of DA content in the striatum and TH-IR in the SNpc induced by icariin in PD mice model. Additionally, icariin treatment alone significantly induced the phosphorylation of Akt and ERK in a time dependent pattern in dopaminergic MES 23.5 cells. These effects were abolished by co-treatment with LY294002 or PD98059. CONCLUSION: These data demonstrated that icariin has neuroprotective effect on dopaminergic neurons in PD mice model and the potential mechanisms might be related to PI3K/Akt and MEK/ERK pathways.

Icariin protects against MPP+-induced neurotoxicity in MES23.5 cells.

Icariin is the major bioactive component of Epimedium and has been demonstrated to be a potential drug for age-related diseases. The present study was aimed to investigate the neuroprotective properties of icariin against 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in MES23.5 cells and the possible mechanisms. MTT assay showed that treatment with MPP+ attenuated the cell viability in a dose-dependent manner in MES23.5 cells. Icariin pretreatment resulted in an enhancement of survival. Immunocytochemistry analysis revealed that icariin treatment attenuated MPP+-induced loss of tyrosine hydroxylase (TH) positive cells. Meanwhile, Western blot confirmed MPP+ significantly decreased the TH protein expression, and icariin pretreatment could reverse the toxic effect of MPP+. Moreover, flow cytometry showed that MPP+-induced decrease of the mitochondrial membrane potential could be partly restored by icariin. Furthermore, real-time RT-PCR and Western blot analysis demonstrated that icariin treatment restored the MPP+-induced up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein expressions. Western blot data also revealed the inhibitory effect of icariin on MPP+-induced up-regulation of cleaved caspase-3. These findings provide the evidence that icariin has neuroprotective properties against MPP+-induced neurotoxicity in MES23.5 cells and the mechanism might be related to the anti-apoptotic action of icariin.

[Chemical Constituents from Jasminum elongatum].

Objective: To investigate the chemical constituents of Jasminum elongatum. Methods: The chemical constituents were isolated and purified by various column chromatographic methods and preparative HPLC. Their structures were identified by physicochemical properties and spectral analysis. Results: Nine compounds were isolated from the methanol extract of Jasminum elongatum and identified as protocatechuic acid( 1),caffeic acid( 2),salicylic acid( 3),isovanillic acid( 4),ferulic acid( 5),methyl caffeate( 6),caffein( 7),3,6-diisopropylpiperazin-2,5-dione( 8),scopoletin( 9). Conclusion: All the compounds are isolated from the this plants for the first time.

[Rapid Identification of Chemical Components in Polygonum multiflorum Formula Granules by UPLC/Q-TOF MS].

OBJECTIVE: To establish a simple and reliable method for rapid separation and identification of chemical components in Polygonum multiflorum Formula Granules. METHODS: An ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method( UPLC/Q-TOF MS) was used. The separation was performed on an Agilent Eclipse Plus C18 RRHD(100 mm x 2.1 mm, 1.8 µm) column with a mobile phase of water and acetonitrile in a gradient elution mode. The flow rate was 0.4 mL/min and the column temperature was maintained at 25 degrees C. TOF MS was applied for qualitative analysis under positive ion mode. RESULTS: Five compounds were identified by the time of flight mass spectrometry and literature data. CONCLUSION: This method is accurate, rapid and sensitive, it can provide reference for the quality control of Polygonum multiflorum Formula Granules.

Pro-inflammatory cytokines in paraventricular nucleus mediate the cardiac sympathetic afferent reflex in hypertension.

Our previous studies showed that pro-inflammatory cytokines (PIC) in the hypothalamic paraventricular nucleus (PVH) potentiated the cardiac sympathetic afferent reflex (CSAR) in normotensive rats. This study determined whether PIC in the PVH mediate enhanced CSAR and over-excited sympathetic activity in spontaneously hypertensive rats (SHR). CSAR was evaluated by renal sympathetic nerve activity (RSNA) response to epicardial application of bradykinin (BK). Inflammatory cytokine levels were measured with ELISA. In both SHR and normotensive Wistar-Kyoto (WKY) rats, PVH microinjection of PIC, tumour necrosis factor (TNF)-α or interleukin (IL)-1ß, increased the baseline mean arterial blood pressure (MAP), RSNA and the CSAR, but anti-inflammatory cytokines (AIC), IL-4 or IL-13, only increased the baseline MAP. PVH pretreatment with PIC caused sub-response dose of angiotension (Ang) II to produce baseline RSNA and MAP elevation and the CSAR enhancement responses, but AIC (IL-4 or IL-13) did not. PVH microinjection of PIC induced greater changes in SHR than in normotensive WKY rats. In addition, stimulation of cardiac sympathetic afferents with epicardial application of BK increased PIC levels in the PVH in both SHR and WKY rats. Intrapericardial administration of resiniferatoxin (RTX) which abolished the CSAR decreased the PIC levels in the PVH to a lower level in SHR than in WKY rats. These results suggest that the increased PIC in the PVH in SHR mediated the increased sympathetic outflow and the enhanced CSAR, and that the augmented effect of Ang II in the PVH on sympathetic activity and the CSAR is also associated with PIC.

Effect of Rac1 downregulation mediated by shRNA on the biological behaviour of human cervical cancer cells.

OBJECTIVE: The function of Ras-related C3 botulinum toxin substrate1 (Rac1) in the progression of cervical cancer is unclear. This study used RNA interference technology to explore the involvement of Rac1 in the regulation of cervical cancer cells. METHODS: A short hairpin (sh) RNA plasmid targeting Rac1 was constructed and transfected into HeLa cells. Rac1 mRNA and protein levels were investigated by reverse transcription-polymerase chain reaction and Western blot, respectively. Cell proliferation and cisplatin chemosensitivity were determined using the methyl thiazolyl tetrazolium assay. The Matrigel™ assay and flow cytometry were used to assess cell invasion and apoptosis, respectively. The concentration of matrix metalloproteinase (MMP)-2 in cell supernatants was detected by enzyme-linked immunosorbent assay. RESULTS: Rac1 expression was significantly downregulated at the mRNA and protein levels in HeLa cells transfected with Rac1 shRNA, and the cell proliferation and invasion capability of cells was decreased. Rac1 downregulation was associated with a decrease in MMP-2 secretion, and increased cell chemosensitivity to cisplatin and cisplatin-induced apoptosis. CONCLUSIONS: Rac1 may play an important role in cervical cancer progression and could be a potential target for anticancer therapy.

Resveratrol improves learning and memory in normally aged mice through microRNA-CREB pathway.

Resveratrol (RSV) is a natural compound found in grapes and red wine. It has been well known for its beneficial effects as a dietary supplement in prevention of cardiovascular diseases and cancer. Recently, in vitro studies have reported the neuroprotective role of RSV in neurodegenerative process in Alzheimer's disease (AD). However, in vivo effects of RSV on the decline of brain function accompanying the aging process, especially those on cognitive loss, have not been not investigated. Here we report that, after intraventricular injection of RSV for one week in 8-9 month-old mice, the long-term memory formation and the LTP induction from hippocampus CA1 were improved. The RSV enhancement effects were blocked in SIRT1 mutant mice. Additional experiments suggest that RSV effects are likely to be mediated through reduced expressions of miR-134 and miR-124, which may in turn up-regulate CREB levels to subsequently promote BDNF synthesis. These findings demonstrate a role for RSV in cognition and a microRNA-CREB-BDNF mechanism by which RSV regulates these processes, demonstrating its value as a potential therapeutic target against CNS disorders in aging.

[Nasal carriage of community-acquired Staphylococcus aureus and drug sensitivity tests in healthy children in Chengdu].

OBJECTIVE: To investigate nasal carriage of community-acquired Staphylococcus aureus and its drug sensitivities in healthy children in Chengdu. METHODS: Nasal swabs were collected from healthy children from primary schools and kindergartens in Chengdu in two stages (2005-2007 and 2008-2010). All specimens were cultivated. Once S. aureus was identified, drug susceptibility tests (disk diffusion method) were performed with penicillin, erythromycin, clindamycin, ceftazidime and vancomycin. RESULTS: 430 S. aureus were identified from 2373 specimens, with a positive rate of 18.12%. Resistant to penicillin was found in 90% of tests. The isolated S. aureus was also resistant (6.28%) to methicillin-resistant Staphylococcus aureus (MRSA). The first stage identified higher rate of MRSA than the second stage (4.28% versus 9.25%, P = 0.037). Isolates from children living in cities were more likely to be resistant to cefoxitin than isolates from children living in countryside (14.74% versus 2.56%, P = 0.006) in the second stage. We did not find vancomycin-resistant S. aureus. CONCLUSION: Nasal carriage of S. aureus among healthy children in Chengdu is common and the nasal carried S. aureus is highly resistant to commonly used antibiotics.

[Expression of Sema3A and NP-1 in spinal cord and spared dorsal root ganglion after partial dorsal root rhizotomy of cat].

OBJECTIVE: To investigate the expression of semaphorin 3A (Sema3A) and neuropilin 1 (NP-1) in spinal cord and dorsal root ganglion after partial dorsal root rhizotomy. METHODS: 15 adult cats were used for this study and divided into 3 groups: normal control group, 7 d and 14 d postoperative groups (7Th day and 14th day groups) undergoing unilateral partial dorsal root rhizotomy. The L3, L5 and L6 segments of spinal cord and L6 dorsal root ganglia (DRG) in operated side were made into frozen sections. By immunohistochemistry ABC method, the sections of spinal cord were stained with specific Sema3A antibody, and L6 DRG were stained with NP-1 antibody. The mean optical density (OD) of Sema3A immunoreactivity in dorsal horn was measured and the number of NP-1 positive medium-small sized neurons in spared DRG was counted. RESULTS: After partial dorsal root rhizotomy, in L3 segment the expression of Sema3A decreased in 7th day group (0.25 +/- 0. 14) compared with that in normal group(0. 37 +/- 0.87) (P < 0.05), but kept the level along to 14th day group (0.27 +/- 0.09); in L5 segment, the expression of Sema3A decrea sed in 7th day group (0.26 +/- 0.11) (P < 0.05), and then recovered to normal level in 14th day group (0.33 +/- 0.09); in L6 segment, OD values in dorsal horn had no changes to all groups. The number of NP-1 positive medium-small sized neurons in spared DRG (30.85 +/- 10.26) was decreased in 7th day group (P < 0.05), compared with that in normal group (45.06 +/- 12.47), while increased in 14th day group (P < 0.05). CONCLUSION: Changes in the expression of Sema3A in spinal cord and the expression of NP-1 in L6 DRG after partial root rhizotomy may be involved in collateral sprouting of spared root in superficial lamina.