Objective To test the hypothesis of autophagy that silencing PHD2 gene could increase hypoxia inducible factor (HIF)-1α levels in the renal medulla and attenuate hypoxia injury in cultured human renal proximal tubular epithelial cell (HK-2) under cobalt dichloride (CoCl2) exposure.Methods HK-2 cells were harvested at hour 0,6,12,24,36 and 48 after exposure to CoC12 (200 μmol/L).The role of HIF/PHD pathway in CoCl2-induced cell apoptosis/autophagy was studied by employing small-interfering RNA (siRNA).Dynamic profiles of apoptosis markers (Bax,Bcl-xl) and autophagy marker (LC3) of HK-2 cells within 48 h after exposing to CoCl2 were recorded.Alamar Blue assay was used for quantitative analysis of cellular growth and viability.Electron microscopy analysis was employed to evaluate the changes in autophagic structures.Results The protein expressions of PHD2 were gradually increased after exposing to CoCl2 (200 μmol/L),with statistics significance at 24 h and reached the peak at 48 h (both P ＜ 0.01).PHD2 siRNA reduced PHD2 levels by ＞ 60％ and significantly increased HIF-1α protein levels (P ＜ 0.01),but had little effect on HIF-2α.The protein expression of Bcl-xl was significantly up-regulated,while the level of Bax and LC3-Ⅱ/LC3-Ⅰ were down-regulated in PHD2 siRNA group (all P ＜ 0.01),compared with the negative control group.Meanwhile,either 3-Methyladenine (an autophagy inhibitor) treatment or PHD2 knockdown rescued cell death and increased cell viability through autophagy inactivation.The ratio of LC3-Ⅱ/LC3-Ⅰ and the quantity of autophagosomes were decreased,and the cell ultrastructure was also relatively intacter than the negative control group.Of interest,co-administration of HIF-1α siRNA with PHD2 siRNA abrogated renoprotective effect conveyed by PHD2 siRNA alone,suggesting that activation of endogenous HIF-1α-dependent pathways mediated the autophagy inactivation effects of PHD2 silencing.Conclusions Direct inhibition of PHD2 promotes renal epithelia cell survival against CoCl2-induced cell apoptosis/autophagy.Activation of the HIF-1α signaling pathway is required to reduce apoptosis and autophagy via up-regulating the expression of Bcl-xl protein.

Objective To investigate the molecular mechanism of protection of ischemia preconditioning on renal ischemia reperfusion injury. Methods Male C57/BL6N mice were randomly divided into two groups: in IR group, 35 min ischemia was induced by occlusion of both renal pedicles followed by 24 h perfusion (I/R). 15 min ischemia was induced 4 days before I/R in IPC group. Blood sample and kidney were collected in IR and IPC group after 24 h perfusion. Serum creatinine (Scr) and histological changes were used to evaluate the renal injury. PHD2 and HIF-1αwere evaluated by Western blotting, miR-21 expression was confirmed by real-time PCR. In vitro, hypoxic model was established by 1% O2 in HK-2 cells. Knockdown of miR-21 in hypoxic model was perfermed by locked nucleic acid modified-anti-miR-21 transfection. The levels of miR-21, HIF-1α and PHD2 mRNA were confirmed by real-time PCR. The levels of HIF-1α and PHD2 proteins were tested by Western blotting. Results In vivo, Compared with IR group, the renal function and histological changes were improved in IPC group (P<0.01). Compared with IR group, the expression of miR-21(P<0.01) and HIF-1α(P<0.05) were increased in IPC group, while PHD2 was reduced (P<0.01). In vitro, hypoxia reduced miR-21. The inhibition of miR-21 could increased the expression of PHD2 (P<0.05). Conclusions Ischemia preconditioning may exert protection against renal ischemia reperfusion injury by inhibiting PHD2.

To analyze the interest distribution relationship and symbiotic mode in the construction of urban medi-cal groups,based on the symbiotic theory,a framework for analyzing the interest distribution relationship of urban medical groups is constructed.Based on actual cases,urban medical groups are summarized and divided into four types:one-way supply type,partial benefit symbiosis type,asymmetric reciprocity symbiosis type,and symmetric reciprocity symbiosis type.The focus of a one-way supply oriented urban medical group is on reshaping the profit distribution mechanism,a partial benefit symbiotic urban medical group is on establishing a profit compensation mechanism for benefit medical institutions,and an asymmetric and mutually beneficial symbiotic urban medical group is on building a long-term benefit balance mechanism.

ObjectiveTo explore the possible mechanism of Chuanxiong （Rhizoma Chuanxiong） & Tianma （Rhizoma Gastrodiae） herbal pair in treating migraines based on AMP-activated protein kinase （AMPK）/transient receptor potential A1 channel （TRPA1） pathway. MethodsForty-eight healthy male SD rats were randomly divided into control group， model group， and Chuanxiong Tianma medication group， with 16 rats in each group. The control group and model group were given 10 ml/kg of normal saline by gavage， while the Chuanxiong Tianma medication group was given 0.675 g/kg of Chuanxiong Tianma herbal pair by gavage， once daily for 8 consecutive days in both groups. Migraime model was performed before the last administration， with subcutaneous injection of 10 ml/kg of normal saline in the control group， and subcutaneous injection of 10 ml/kg of nitroglycerin in the model group and Chuanxiong Tianma medication group. The Von Frey filament was used to measure the periorbital mechanical pain threshold of rats. The enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of calcitonin gene-related peptide （CGRP） in rat serum and cerebrospinal fluid. The nitric oxide （NO） assay kit was used to determine the NO level in serum and cerebrospinal fluid. RT-PCR was usedto detect the mRNA expression levels of immediate-early genes in the trigeminal ganglion of rats （c-Fos）， CGRP， transient receptor potential V1 channel （TRPV1）， AMPK alpha subunit （PRKAA）， and TRPA1. Immunofluorescence was used to detect the number of c-Fos-positive cells in the trigeminal cervical complex （TCC） and the protein expression levels of phosphorylated AMPK （pAMPK） and TRPA1 in the trigeminal ganglion. ResultsCompared to those in the control group， the mechanical stimulation threshold and pAMPK protein expression in the model group decreased， while the levels of CGRP and NO in serum， c-Fos， CGRP， TRPV1 and TRPA1 mRNA levels in the trigeminal ganglion， TRPA1 protein expression， and the number of c-Fos-positive cells in the TCC significantly increased （P＜0.05）. Compared to those in the model group， the mechanical stimulation threshold and pAMPK protein expression in the Chuanxiong Tianma medication group significantly increased， while the levels of CGRP and NO in serum， c-Fos， CGRP， TRPV1 and TRPA1 mRNA levels in the trigeminal ganglion， TRPA1 protein expression， and the number of c-Fos-positive cells in the TCC significantly decreased （P＜0.05）. ConclusionChuanxiong Tianma herbal pair may improve migraine symptoms by regulating the AMPK/TRPA1 pathway in the trigeminal ganglion and increasing the mechanical pain threshold.

Objective To investigate the effect of Yipi Yanggan prescription on the malignant transformation of liver stem cells in liver precancerous lesion induced by diethylnitrosamine (DEN) and its possible molecular mechanism. Methods A total of 35 male Sprague-Dawley rats were randomly divided into normal control group (blank group), DEN model group (model group), DEN+Yipi Yanggan prescription group (Yipi Yanggan prescription group), and DEN+Hugan tablet group (Hugan tablet group), with 5 rats in the blank group and 10 rats in the other three groups. Intraperitoneal injection of DEN was performed to establish a model of liver precancerous lesion, the rats were sacrificed after 16 weeks of administration. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Alb) were measured; liver tissue was collected to observe the changes in size and appearance and calculate liver weight ratio (liver index); HE staining and Sirius Red staining were used to observe the pathological and morphological changes of rat liver tissue; immunohistochemistry was used to measure the expression of OV6 and glutathione S-transferase-Pi (GST-Pi); RT-PCR was used to measure the mRNA expression of EpCAM, CD133, and CD90, and Western blot was used to measure the protein expression of PI3K, Akt, and mTOR and their phosphorylation level. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the model group, the Yipi Yanggan prescription group and the Hugan tablet group had significant improvements in liver pathology and morphology, significant reductions in liver index and the levels of ALT and AST, and a significant increase in the level of Alb (all P < 0.05), as well as significant reductions in the protein expression levels of GST-Pi, OV6, p-PI3K, p-Akt, and p-mTOR and the mRNA expression levels of EpCAM, CD133, and CD90 (all P < 0.05). Compared with the Hugan tablet group, the Yipi Yanggan prescription group showed a more significant protective effect on the liver, with significant reductions in liver index and the levels of ALT and AST, and a significant increase in the level of Alb (all P < 0.05), as well as significant reductions in the protein expression levels of GST-Pi, OV6, p-PI3K, p-Akt, and p-mTOR and the mRNA expression levels of EpCAM, CD133, and CD90 (all P < 0.05). Conclusion Yipi Yanggan prescription can improve liver precancerous lesion induced by DEN in rats by inhibiting the malignant transformation of liver stem cells, and its mechanism of action may be associated with the PI3K/Akt/mTOR signaling pathway.

Postnatal heart maturation is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine. While static snapshots of the maturing heart have provided much insight into its molecular signatures, few key events during postnatal cardiomyocyte maturation have been uncovered. Here, we report that cardiomyocytes (CMs) experience epigenetic and transcriptional decline of cardiac gene expression immediately after birth, leading to a transition state of CMs at postnatal day 7 (P7) that was essential for CM subtype specification during heart maturation. Large-scale single-cell analysis and genetic lineage tracing confirm the presence of transition state CMs at P7 bridging immature state and mature states. Silencing of key transcription factor JUN in P1-hearts significantly repressed CM transition, resulting in perturbed CM subtype proportions and reduced cardiac function in mature hearts. In addition, transplantation of P7-CMs into infarcted hearts exhibited cardiac repair potential superior to P1-CMs. Collectively, our data uncover CM state transition as a key event in postnatal heart maturation, which not only provides insights into molecular foundations of heart maturation, but also opens an avenue for manipulation of cardiomyocyte fate in disease and regenerative medicine.
Gene Expression Regulation ; Heart ; Myocytes, Cardiac/metabolism* ; Single-Cell Analysis ; Transcription Factors/metabolism*