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Introduction: As a new discipline, network pharmacology has been widely used to disclose the material basis and mechanism of Traditional Chinese Medicine in recent years. However, numerous researches indicated that the material basis of TCMs identified based on network pharmacology was the mixtures of beneficial and harmful substances rather than the real material basis. In this work, taking the anti-NAFLD (non-alcoholic fatty liver disease) effect of Bai Shao (BS) as a case, we attempted to propose a novel bioinformatics strategy to uncover the material basis and mechanism of TCMs in a precise manner. Methods: In our previous studies, we have done a lot work to explore TCM-induced hepatoprotection. Here, by integrating our previous studies, we developed a novel computational pharmacology method to identify hepatoprotective ingredients from TCMs. Then the developed method was used to discover the material basis and mechanism of Bai Shao against Non-alcoholic fatty liver disease by combining with the techniques of molecular network, microarray data analysis, molecular docking, and molecular dynamics simulation. Finally, literature verification method was utilized to validate the findings. Results: A total of 12 ingredients were found to be associated with the anti-NAFLD effect of BS, including monoterpene glucosides, flavonoids, triterpenes, and phenolic acids. Further analysis found that IL1-ß, IL6, and JUN would be the key targets. Interestingly, molecular docking and molecular dynamics simulation analysis showed that there indeed existed strong and stable binding affinity between the active ingredients and the key targets. In addition, a total of 23 NAFLD-related KEGG pathways were enriched. The major biological processes involved by these pathways including inflammation, apoptosis, lipid metabolism, and glucose metabolism. Of note, there was a great deal of evidence available in the literature to support the findings mentioned above, indicating that our method was reliable. Discussion: In summary, the contributions of this work can be summarized as two aspects as follows. Firstly, we systematically elucidated the material basis and mechanism of BS against NAFLD from multiple perspectives. These findings further enhanced the theoretical foundation of BS on NAFLD. Secondly, a novel computational pharmacology research strategy was proposed, which would assist network pharmacology to uncover the scientific connotation TCMs in a more precise manner.

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The efforts focused on discovering potential hepatoprotective drugs are critical for relieving the burdens caused by liver diseases. Traditional Chinese medicine (TCM) is an important resource for discovering hepatoprotective agents. Currently, there are hundreds of hepatoprotective products derived from TCM available in the literature, providing crucial clues to discover novel potential hepatoprotectants from TCMs based on predictive research. In the current study, a large-scale dataset focused on TCM-induced hepatoprotection was established, including 676 hepatoprotective ingredients and 205 hepatoprotective TCMs. Then, a comprehensive analysis based on the structure-activity relationship, molecular network, and machine learning techniques was performed at molecular and holistic TCM levels, respectively. As a result, we developed an in silico model for predicting the hepatoprotective activity of ingredients derived from TCMs, in which the accuracy exceeded 85%. In addition, we originally proposed a material basis and a drug property-based approach to identify potential hepatoprotective TCMs. Consequently, a total of 12 TCMs were predicted to hold potential hepatoprotective activity, nine of which have been proven to be beneficial to the liver in previous publications. The high rate of consistency between our predictive results and the literature reports demonstrated that our methods were technically sound and reliable. In summary, systematical predictive research focused on the hepatoprotection of TCM was conducted in this work, which would not only assist screening of potential hepatoprotectants from TCMs but also provide a novel research mode for discovering the potential activities of TCMs.

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Background: Rheumatoid arthritis (RA) is the most common inflammatory arthropathy. Immune dysregulation was implicated in the pathogenesis of RA. Thus, the aim of the research was to determine the immune related biomarkers in RA. Methods: We downloaded the gene expression data of RA in GSE89408 and GSE45291 from Gene Expression Omnibus public database (GEO). Differentially expressed genes (DEGs) were identified between RA and control groups. Infiltrating immune cells related genes were obtained by ssGSEA and weighted gene co-expression network analysis (WGCNA). We performed functional enrichment analysis of differentially expressed immunity-related genes (DEIRGs) by "clusterProfiler" R package, key genes screening by protein-protein interaction (PPI) network of DEIRGs. And mice collagen-induced arthritis (CIA) model was employed to verify these key genes. Results: A total of 1,885 up-regulated and 1,899 down-regulated DEGs were identified in RA samples. The ssGSEA analysis showed that the infiltration of 25 cells was significantly different. 603 immune related genes were obtained by WGCNA, and 270 DEIRGs were obtained by taking the intersection of DEGs and immune related genes. Enrichment analyses indicated that DEIRGs were associated with immunity related biological processes. 4 candidate biomarkers (CCR7, KLRK1, TIGIT and SLAMF1) were identified from the PPI network of DEIRGs and literature research.In mice CIA model, the immunohistochemical stain showed SLAMF1 has a significantly high expression in diseased joints. And flow cytometry analysis shows the expression of SLAMF1 on CIA mice-derived CTL cells, Th, NK cells, NKT cells, classical dendritic cell (cDCs) and monocytes/macrophages was also significantly higher than corresponding immune cells from HC mice. Conclusion: Our study identified SMLAF1 as a key biomarker in the development and progression of RA, which might provide new insight for exploring the pathogenesis of RA.

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Transient receptor potential vanilloid type 4 (TRPV4) can function as an oncogene or tumor suppressor depending on the tumor types. However, little is known regarding the effect of TRPV4 in nasopharyngeal carcinoma (NPC), a highly prevalent malignancy in Southern China and Southeast Asia. We found that TRPV4 mRNA and protein levels were significantly upregulated in NPC tissues. In addition, activation of TRPV4 in NPC cell lines using GSK1016790A (100 nM) induced a Ca2+ influx, whereas pharmacological inhibition or gene knockdown of TRPV4 reduced the proliferation rates of NPC cells. TRPV4 knockdown also decreased the growth of tumor xenografts in vivo. Mechanistically, TRPV4-mediated tumorigenesis is dependent on the activation of Ca2+/calcineurin/calcineurin-nuclear factor of activated T cell 4 (NFAT4) signaling. Furthermore, NFAT4 protein level was overexpressed in NPC tissues and correlated positively with TRPV4. Taken together, TRPV4 promotes the malignant potential of NPC cells by activating NFAT4 signaling. Our findings highlight TRPV4-NFAT4 axis as a potential therapeutic target in NPC.

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Luteolin attenuates myocardial ischemia/reperfusion (I/R) injury in diabetes through activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidative response. Though sestrin2, a highly conserved stress-inducible protein, is regarded as a modulator of Nrf2 and reduces I/R injury, the effect of sestrin2 on luteolin-induced prevention of the diabetic heart from I/R injury remains unclear. We hypothesized that luteolin could relieve myocardial I/R injury in diabetes by activating the sestrin2-modulated Nrf2 antioxidative response. Diabetes was induced in rats using a single dose of streptozotocin (65 mg kg-1, i.p.) for 6 weeks, and then luteolin (100 mg kg-1 d-1, i.g.), Nrf2 inhibitor brusatol, or sestrin2 blocker leucine was administered for 2 consecutive weeks. After that, the hearts were isolated and exposed to global I/R (30 min/120 min). Luteolin markedly improved cardiac function, myocardial viability and expressions of Nrf2-regulated antioxidative genes, and reduced lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R hearts. Ca2+-induced mitochondrial permeability transition and membrane potential disruption were markedly inhibited in luteolin-treated diabetic ventricular myocytes. All these effects of luteolin were significantly reversed by Nrf2 inhibitor brusatol or sestrin2 inhibitor leucine. Luteolin-induced diminished Keap1 and augmented nuclear translocation and ARE binding activity of Nrf2 were hampered by leucine in the diabetic I/R heart. In addition, luteolin-induced augmented transcription of sestrin2 was markedly blocked by brusatol in the diabetic I/R heart. These data suggest that sestrin2 and Nrf2 positively interact to promote antioxidative actions and attenuate mitochondrial damage, by which luteolin relieves diabetic myocardial I/R injury.

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Macrophages can change their physiology in response to microenvironmental signals. This differentiation into classically activated M1 or alternatively activated M2 macrophages is known as polarization. In this study, we isolated bone marrow-derived macrophages from ß2m-deficient (deficient in both MHC class Ia and Ib) and Kb Db -deficient (deficient only in MHC class Ia) mice and found that ß2m-deficient macrophages showed a significantly lower M2b polarization efficiency. In addition, the absence of constitutive MHC class Ib expression decreased the stability of the Notch-1 intracellular domain. Finally, we found that ß2m-deficient mice exposed to irradiation showed reduced bacterial translocation and sepsis severity. Overall, our study demonstrates that MHC class Ib molecules are essential for M2b macrophage polarization and suggests that MHC class Ib molecules play an important role during infection-induced innate immunity.

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Medical parasitology education has been facing some difficulties, because it is a course of wide range, lacking clinical cases and concerned specimens of parasites currently. In addition, its relationship with life is not closely enough. All these reasons may impact the effect of class education negatively. Therefore, it is important to increase the vitality of parasitology education and diversify the instructional mode by using the resources from Internet. In recent years, the Discovery Channel has uploaded a documentary Monsters Inside Me online. This documentary is high professional and closely linked with parasitology. It maintains numbers of clinical cases about parasitic diseases. Each episode is about 3 minutes and shortly enough to be introduced into class teaching. However, this resource has not been fully used in domestic temporally. We found that direct introduction of the documentary into class teaching can enrich teaching forms to attract learning interest of students, and finally improve the teaching effect of class. Above that, another popular documentary A Bite of China involves many related knowledge points of parasitology. The appropriate usage of the knowledge can build up close linkage between book and life, which is extremely helpful to give students a deep impression of parasitology. In brief, it is our strong recommendation to introduce the documentary Monsters Inside Me into class.

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BACKGROUND: A hallmark of atherosclerosis is progressive intimal thickening (namely neointimal hyperplasia), which leads to occlusive vascular diseases. Over-production of reactive oxygen species (ROS) and alteration of Ca2+ signaling are among the key factors contributing to neointimal growth. In the present study, we investigated the role of TRPM2, a ROS-sensitive Ca2+ entry channel, in neointimal hyperplasia. METHODS AND RESULTS: Perivascular cuffs were used to induce neointimal hyperplasia in rat/mouse arteries. Immunostaining showed numerous TRPM2-positive smooth muscle cells in neointimal regions. ROS were over-produced and PCNA-positive proliferating cells were numerous in the neointimal regions. The neointimal hyperplasia was substantially reduced in Trpm2 knockout mice compared with wild-type mice. In the cultured rat/mouse aortic smooth muscle cells, H2O2 treatment was found to stimulate cell proliferation and migration. The effect of H2O2 was reduced by a TRPM2-specific blocking antibody TM2E3 or Trpm2 knockout. The signaling molecules downstream of TRPM2 were found to be Axl and Akt. CONCLUSIONS: These data suggest a critical functional role of TRPM2 in the progression of neointimal hyperplasia. The study also highlights the possibility of targeting TRPM2 as a potential therapeutic option for the treatment of occlusive vascular diseases.

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Transient receptor potential (TRP) superfamily is a superfamily of cation channels that can be divided into seven subfamilies. TRPM2 is the second member of the TRPM subfamily, which includes eight members, namely TRPM1-8. TRPM2 is widely expressed in excitable and non-excitable cells, where it forms a Ca(2+)-permeable cation channel and performs diverse cellular functions. TRPM2 channels are activated by ADP-ribose (ADPR), Ca(2+), H2O2 and other reactive oxygen species (ROS). It is established that TRPM2 serves as a cellular sensor for oxidative stress, mediating oxidative stress-induced [Ca(2+)]i increase and contributing to pathological processes in many cell types. Accumulating evidence has indicated that TRPM2 is a potential therapeutic target for oxidative stress-related diseases. This review will highlight recent progress in this field.

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Luteolin is an antioxidative, antitumor and anti-inflammatory flavone. It has been shown to reduce endothelial dysfunction, but the mechanism is not clear. We set out to explore the effects of luteolin on apoptosis and its mechanism of action in endothelial cells. The effect of luteolin on pyrogallol-induced superoxide stress and the subsequent apoptosis was studied in the mouse heart capillary endothelial cell line H5V and human umbilical vein endothelial cells, by the use of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Hoechst staining, and western blot. Pyrogallol (0-400 µm) dose-dependently induced reactive oxygen species production, cytotoxicity, an annexin V-fluorescein isothiocyanate increase, mitochondrial transmembrane depolarization and DNA condensation in both H5V and human umbilical vein endothelial cells; these actions were reversed by luteolin (0.78-50 µm) in a concentration-dependent manner. Luteolin suppressed the poly (ADP-ribose) polymerase activation, caspase-8 cleavage and p38 mitogen-activated protein kinase activation triggered by pyrogallol, and stimulated the extracellular signal-regulated kinase signaling pathway to counteract the pyrogallol-induced apoptotic signals. Luteolin is an effective agent for the protection of endothelial cells from superoxide stress-induced apoptosis via the extracellular signal-regulated kinase signaling pathway.

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ETHNOPHARMACOLOGICAL RELEVANCE: Total flavonoids of Flos Chrysanthemi (TFFC) are known to modulate vascular functions, but their effect on endothelial cells injured by oxidative stress is unknown. Our objective was to investigate the vasoprotective effect and mechanism of action of TFFC on rat mesenteric artery exposed to superoxide anions produced by pyrogallol. MATERIALS AND METHODS: The vasoprotective effect and mechanism of action of TFFC on primary cultured rat mesenteric arterial endothelial cells and small mesenteric arteries was investigated using small-vessel myography, fluorescent Ca(2+) measurement, fluorescent membrane potential measurement and oxidative fluorescent studies. RESULTS: Experiments using small-vessel myography of third-order rat mesenteric arterial rings showed that pretreatment with pyrogallol (10-1000µM), an auto-oxidizing source of superoxide anions, dose-dependently decreased ACh-induced endothelium-dependent relaxation. TFFC (2.5-320mg/L) evoked a concentration-dependent dilation (pD(2): 29.6±0.276mg/L), which was weakened by ChTX plus apamin. TFFC markedly attenuated the inhibition of vasorelaxation induced by pyrogallol (E(max) elevated from 50.4±7.36% to 86.2±3.61%, and pD(2) increased from 6.74±0.06 to 7.28±0.12). Furthermore, in primary cultured endothelial cells, fluorescent Ca(2+) measurement, fluorescent membrane potential measurement and oxidative fluorescent studies demonstrated that ACh-induced endothelial Ca(2+) influx and hyperpolarization were significantly weakened by the increased basal superoxide level induced by pyrogallol. When the endothelial cells were concurrently exposed to TFFC, the impairment effect of oxidative stress on ACh-induced Ca(2+) influx, hyperpolarization and vasorelaxation were attenuated due to its superoxide-lowering activity. CONCLUSION: This study shows that oxidative stress has a pronounced deleterious effect on EDHF-mediated vasorelaxation to ACh in rat mesenteric artery. TFFC has vasodilating effect and protects EDHF-mediated vasodilator reactivity from oxidative stress. Thus, our experiments suggest that TFFC is potentially useful for the development of therapeutic treatments for cardiovascular diseases associated with oxidative stress.

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BACKGROUND AND PURPOSE: Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+) -permeable channel with multiple modes of activation. Apigenin is a plant-derived flavone, which has potential preventive effects on the development of cardiovascular disease. We set out to explore the effects of apigenin on TRPV4 channel activity and its role in vasodilatation. EXPERIMENTAL APPROACH: The effects of apigenin (0.01-30 µM) on TPRV4 channels were investigated in HEK293 cells over-expressing TRPV4, rat primary cultured mesenteric artery endothelial cells (MAECs) and isolated small mesenteric arterial segments using whole-cell patch clamp, fluorescent Ca(2+) imaging, intracellular recording and pressure myography. KEY RESULTS: Whole-cell patch clamp and fluorescent Ca(2+) imaging in HEK cells over-expressing TRPV4 showed that apigenin concentration-dependently stimulated the TRPV4-mediated cation current and Ca(2+) influx. In MAECs, apigenin stimulated Ca(2+) influx in a concentration-dependent manner. These increases in cation current and Ca(2+) influx were markedly inhibited by TRPV4-specific blockers and siRNAs. Furthermore, pressure myography and intracellular recording in small third-order mesenteric arteries showed that apigenin dose-dependently evoked smooth muscle cell membrane hyperpolarization and subsequent vascular dilatation, which were significantly inhibited by TRPV4-specific blockers. TRPV4 blocker or charybdotoxin (200 nM) plus apamin (100 nM) diminished the apigenin-induced dilatation. CONCLUSION AND IMPLICATIONS: This is the first study to demonstrate the selective stimulation of TRPV4 by apigenin. Apigenin was found to activate TRPV4 channels in a dose-dependent manner in HEK cells over-expressing TRPV4 and in native endothelial cells. In rat small mesenteric arteries, apigenin acts on TRPV4 in endothelial cells to induce EDHF-mediated vascular dilatation.

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OBJECTIVE: To investigate the effect of bicyclol on vascular oxidative stress injury induced by superoxide anion. METHODS: Rat thoracic aortic rings were isolated for isometric tension recording using organ bath technique. Superoxide arterial injury was induced by pyrogallol exposure, and the effect of bicyclol on endothelium-dependent relaxation was evaluated. RESULTS: Bicyclol (10(-8) - 10(-5) mol/L) relaxed endothelium-intact aortic rings precontracted by phenylephrine. This effect was abolished by L-NAME, an inhibitor of nitric oxide synthase and indomethacin, an inhibitor of cyclooxygenase. Exposure to pyrogallol (500 micromol/L) resulted in decrease of acetylcholine(ACh)-induced endothelium-dependent relaxation in aortic rings, and pre-incubation of bicyclol (10(-5) mol/L) for 45 min improved the relaxation attenuated by pyrogallol. In aortic rings pre-treated with indomethacin, bicyclol increased the ACh-induced relaxation that was inhibited by pyrogallol (500 micromol/L). This effect was not found in aortic rings pre-treated with L-NAME. CONCLUSION: Bicyclol has endothelium-dependent vasodilating effect on rat thoracic aorta and improves vascular function by attenuating oxidative stress. Nitric oxide from endothelium is involved in the anti-oxidative effect of bicyclol.

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OBJECTIVE: To investigate the myocardial electrophysiological effect and its underlying mechanisms of atorvastatin (Ator) on isolated rat hearts injured by ischemia/reperfusion (I/R). METHODS: Isolated SD rat hearts were mounted on Langendorff system, and a local I/R was induced by ligation (30 min) and release (15 min) of the left anterior descending artery. During the reperfusion period, the effect of Ator on diastolic excitation threshold (DET), effective refractory period (ERP) and ventricular fibrillation threshold (VFT) on rat heart were measured. RESULT: Compared with the control group, medium concentration of Ator prolonged the ERP in normal rat hearts; low, medium and high concentration of Ator significantly inhibited the decrease of DET, ERP and VFT induced by I/R. However, pretreatment with L-NAME cancelled these cardiac electrophysiological effects of Ator. CONCLUSION: Ator reduced electrophysiological alteration induced by I/R in isolated rat hearts, which may be mediated by activating nitric oxide pathway to enhance the myocardial electrophysiological stability.

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Nonreceptor tyrosine kinases have an increasingly appreciated role in cardiac injury and protection. To investigate novel tasks for members of the Tec family of nonreceptor tyrosine kinases in cardiac phenotype, we examined the behavior of the Tec isoform in myocardial ischemic injury. Ischemia-reperfusion, but not cardiac protective agents, induced altered intracellular localization of Tec, highlighting distinct actions of this protein compared with other isoforms, such as Bmx, in the same model. Tec is abundantly expressed in cardiac myocytes and assumes a diffuse intracellular localization under basal conditions but is recruited to striated structures upon various stimuli, including ATP. To characterize Tec signaling targets in vivo, we performed an exhaustive proteomic analysis of Tec-binding partners. These experiments expand the role of the Tec family in the heart, identifying the Tec isoform as an ischemic injury-induced isoform, and map the subproteome of its interactors in isolated cells.

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AIMS: The aim of this study was to investigate the effect of alcohol on rat artery and its underlying mechanism. METHODS: The tension of isolated Sprague-Dawley rat thoracic aortic rings and the pressure of rat mesenteric arterial beds perfused with different concentrations of alcohol (0.1-7.0 per thousand) were measured. RESULTS: At resting tensions, alcohol caused a concentration-dependent relaxation on endothelium-denuded aortic rings precontracted with KCl (6 x 10(-2) mol/L) or phenylephrine (PE, 10(-6) mol/L), and this effect was most evident on rings at a resting tension of 3 g. Alcohol induced much less vasodilation on endothelium-intact rings. Alcohol inhibited the CaCl(2)-induced contraction of endothelium-denuded aortic rings precontracted with KCl or PE. Incubation of rings with dantrolene (5 x 10(-5) mol/L), a ryanodine receptor blocker, or 2-aminoethyl diphenylborinate (7.5 x 10(-5) mol/L), an IP(3) receptor blocker, attenuated the vasodilating effect of alcohol on rings precontracted with PE. Alcohol also concentration-dependently relaxed rat mesenteric arterial beds precontracted with KCl (6 x 10(-2) mol/L) or PE (10(-5) mol/L), which was more potent on endothelium-denuded than on endothelium-intact beds. CONCLUSIONS: Alcohol has a vasodilating effect on rat artery depending on the resting tension. Both extracellular and intracellular Ca(2+) mobilization of vascular smooth muscle cells are involved in the vascular effect of alcohol.

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AIM: To investigate the vasodilating effect and its mechanism of ethanol on isolated rat thoracic aorta at different resting tension. METHODS: The tension of the isolated Sprague-Dawley rat thoracic aorta rings perfused with different concentrations of ethanol was measured using organ bath technique. RESULTS: At different resting tension (1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 g), ethanol (0.1-7.0 per thousand) caused a concentration-dependent relaxation on endothelium-denuded aortic rings precontracted with KCl (6 x 10(-2)mol/L) or phenylephrine (PE, 10(-6) mol/L), and the vasodilating effect was the most potent when the aortic rings were at the resting tension of 3 g. Ethanol had much less vasodilating effect on endothelium-intact aortic rings. Ethanol at 3 per thousand (the maximum-effect concentration) inhibited the CaCl2 induced contraction and downward shifted concentration-response curve of endothelium-denuded aortic rings pre-contracted with KCI or PE at the resting tension of 3 g. Incubation of aorta with ruthenium red (10(-5) mol/L) or heparin (50 mg/L) decreased the vasodilating effect of ethanol (3.0 per thousand) on endothelium-denuded aorta precontracted with PE at the resting tension of 3 g. CONCLUSION: Ethanol induces endothelium-independent relaxation on rat thoracic aorta, which is concerned with the resting tension. This effect of ethanol may be mediated by the inhibition of voltage-dependent and receptor-operated Ca2+ channels in the vascular smooth muscle cells. The inhibition of the ryanodine receptor and trisphosphate inositol (IP3) pathway may also contribute to this effect.

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