The intervention effect of Tibetan medicine Siwei Huangqi powder on hypoxic pulmonary hypertension through autophagy signaling pathway induced by AMPK protein / 中国药理学通报

Aim To explore the mechanism of Tibetan medicine Siwei Huangqi powder(SW)in reducing hypoxic pulmonary hypertension. Methods A total of 110 Wistar rats were randomly divided into normoxia control group,hypoxic control group and hypoxic drug group. The two hypoxic groups were divided into 1,3,7,15 and 30 day group according to the exposure time of hypoxic,10 groups in total. The normoxia control group was placed in the atmospheric environment at an altitude of 2 260 meters without intervention; 10 hypoxic groups were placed in a hypobaric hypoxic chamber with a simulated altitude of 5 000 meters. The hypoxic drug group was given SW suspension(0.42 g/100 g)by gavage,and the hypoxic control group was given normal saline by gavage,once a day. The Ppa and RV/(LV+S)were measured at the corresponding time points in each group; the levels of p-AMPK,ULK-1 and LC3 /LC3 Ⅱ protein in lung tissues were measured by WB method. Results Compared with normoxia control group,the ratio of PA and RV/(LV + s)in hypoxic control group increased gradually with the extension of hypoxic exposure time,which was consistent with the thickness of pulmonary artery smooth muscle layer and the changes of pulmonary tissue subcellular organelles. The expression level of p-AMPK protein in lung tissues was also slightly up-regulated(P<0.05),and ULK-1 and LC3 Ⅱ were significantly up-regulated(P<0.01),especially in acute hypoxic. Compared with the hypoxic control group,the increase of Ppa and the thickening of pulmonary artery smooth muscle layer in the hypoxic drug group were significantly reduced(P<0.050.01),while the expression levels of p-AMPK,ULK-1 and LC3 Ⅱ proteins in lung tissues increased with the extension of hypoxic exposure time(P<0.050.001),especially in chronic hypoxic. Conclusion SW can inhibit hypoxic pulmonary hypertension by up-regulating AMPK autophagy signaling pathway.

Adaptive Changes in Hemoglobin Function in High-Altitude Tibetan Canids Were Derived via Gene Conversion and Introgression.

A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb-O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the ß-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked ß-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the ß-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb-O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed's relatively short duration of residency at high altitude.

Transcriptomic profiling reveals gene expression kinetics in patients with hypoxia and high altitude pulmonary edema.

OBJECTIVE: High altitude pulmonary edema (HAPE) is a life threatening condition occurring in otherwise healthy individuals who rapidly ascend to high altitude. However, the molecular mechanisms of its pathophysiology are not well understood. The objective of this study is to evaluate differential gene expression in patients with HAPE during acute illness and subsequent recovery. METHODS: Twenty-one individuals who ascended to an altitude of 3780 m were studied, including 12 patients who developed HAPE and 9 matched controls without HAPE. Whole-blood samples were collected during acute illness and subsequent recovery for analysis of the expression of hypoxia-related genes, and physiologic and laboratory parameters, including mean pulmonary arterial pressure (mPAP), heart rate, blood pressure, and arterial oxygen saturation (SpO2), were also measured. RESULTS: Compared with control subjects, numerous hypoxia-related genes were up-regulated in patients with acute HAPE. Gene network analyses suggested that HIF-1α played a central role in acute HAPE by affecting a variety of hypoxia-related genes, including BNIP3L, VEGFA, ANGPTL4 and EGLN1. Transcriptomic profiling revealed the expression of most HAPE-induced genes was restored to a normal level during the recovery phase except some key hypoxia response factors, such asBNIP3L, EGR1, MMP9 and VEGF, which remained persistently elevated. CONCLUSIONS: Differential expression analysis of hypoxia-related genes revealed distinct molecular signatures of HAPE during acute and recovery phases. This study may help us to better understand HAPE pathogenesis and putative targets for further investigation and therapeutic intervention.

Effects of siRNA on EPAS1 activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rats / 解放军医学杂志

Objective This study explored whether EPAS1 gene is involved in the proliferation of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EPAS1 gene expression was interfered by small interfering RNA (siRNA).Methods The rat primary pulmonary artery smooth muscle cells were cultured and identified by immunofluorescence.The specific lipidosomes of EPAS1 siRNA were constructed and transfected into PASMCs,and the best targets were selected from the three interfering targets.The transfected PASMCs were cultured in hypoxia (37℃,5％ CO2,2％ O2) or normoxia (37℃,5％ CO2,20％ O2) for 24h,48h and 72h,respectively.The PASMCs proliferation was detected by CCK-8 assay.The protein expression of EPAS 1 and vascular endothelial growth factor (VEGF) were determined by Western blotting to investigate the effect of silencing EPAS1 gene expression on the proliferation of PASMCs under hypoxic condition.Results The specific liposomes ofEPAS1 siRNA were successfully constructed and transfected into PASMCs,and the best interfering target were selected from the three interference targets.The proliferation of PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition.Under hypoxic or normoxic condition,PASMCs proliferation and VEGF protein expression of PASMCs were suppressed by EPAS 1 siRNA.Conclusion EPAS 1 gene might be involved in the proliferation of rat PASMCs by regulating VEGF protein level under hypoxic condition.

Shared and unique signals of high-altitude adaptation in geographically distinct Tibetan populations.

Recent studies have used a variety of analytical methods to identify genes targeted by selection in high-altitude populations located throughout the Tibetan Plateau. Despite differences in analytic strategies and sample location, hypoxia-related genes, including EPAS1 and EGLN1, were identified in multiple studies. By applying the same analytic methods to genome-wide SNP information used in our previous study of a Tibetan population (n = 31) from the township of Maduo, located in the northeastern corner of the Qinghai-Tibetan Plateau (4200 m), we have identified common targets of natural selection in a second geographically and linguistically distinct Tibetan population (n = 46) in the Tuo Tuo River township (4500 m). Our analyses provide evidence for natural selection based on iHS and XP-EHH signals in both populations at the p<0.02 significance level for EPAS1, EGLN1, HMOX2, and CYP17A1 and for PKLR, HFE, and HBB and HBG2, which have also been reported in other studies. We highlight differences (i.e., stratification and admixture) in the two distinct Tibetan groups examined here and report selection candidate genes common to both groups. These findings should be considered in the prioritization of selection candidate genes in future genetic studies in Tibet.

Genomic analysis of natural selection and phenotypic variation in high-altitude mongolians.

Deedu (DU) Mongolians, who migrated from the Mongolian steppes to the Qinghai-Tibetan Plateau approximately 500 years ago, are challenged by environmental conditions similar to native Tibetan highlanders. Identification of adaptive genetic factors in this population could provide insight into coordinated physiological responses to this environment. Here we examine genomic and phenotypic variation in this unique population and present the first complete analysis of a Mongolian whole-genome sequence. High-density SNP array data demonstrate that DU Mongolians share genetic ancestry with other Mongolian as well as Tibetan populations, specifically in genomic regions related with adaptation to high altitude. Several selection candidate genes identified in DU Mongolians are shared with other Asian groups (e.g., EDAR), neighboring Tibetan populations (including high-altitude candidates EPAS1, PKLR, and CYP2E1), as well as genes previously hypothesized to be associated with metabolic adaptation (e.g., PPARG). Hemoglobin concentration, a trait associated with high-altitude adaptation in Tibetans, is at an intermediate level in DU Mongolians compared to Tibetans and Han Chinese at comparable altitude. Whole-genome sequence from a DU Mongolian (Tianjiao1) shows that about 2% of the genomic variants, including more than 300 protein-coding changes, are specific to this individual. Our analyses of DU Mongolians and the first Mongolian genome provide valuable insight into genetic adaptation to extreme environments.

No relation between ACE-I/D polymorphism and high altitude pulmonary edema in the Han Chinese / 中国应用生理学杂志

<p><b>OBJECTIVES</b>To explore whether the angiotensin I -converting enzyme (ACE) I/D (insertion/ deletion) polymorphism is associated with the susceptibility to high altitude pulmonary edema (HAPE) in the Han Chinese.</p><p><b>METHODS</b>One hundred and forty-seven HAPE-p (HAPE patients) and 193 HAPE-r (HAPE resistants) were enrolled from the Yushu earthquake reconstruction workers in Qinghai province where the altitude is over 3 500 m above sea level. Blood samples were collected from each of the HAPE-p and HAPE-r groups. Information about physiological phenotypes was obtained via fieldwork investigation. The ACE-I/D polymorphism in HAPE-p and HAPE-r was detected by polymerase chain reaction (PCR).</p><p><b>RESULTS</b>The SaO2 was significantly lower while HR was significantly higher in HAPE-p group than those in HAPE-r group. The genotype frequencies of ACE-I/D for II, ID, DD in HAPE-r and HAPE-p groups were 0.430, 0.446, 0.124 and 0.435, 0.469, 0.095, respectively, the allelic frequencies of I and D were 0.650, 0.350 and 0.670, 0.330, respectively. The OR of ID, DD and D alleles relative to II for HAPE was 0.961 (0.610-1.514), 1.322 (0.634-2.758) and 1.080 (0.783-1.489). There was no significant difference of the genotypic and the allelic frequencies in ACE-I/D polymorphism between HAPE-p and HAPE-r groups.</p><p><b>CONCLUSIONS</b>There is no relation between ACE-I/D polymorphism and HAPE in the Han Chinese.</p>

Metabolic insight into mechanisms of high-altitude adaptation in Tibetans.

Recent studies have identified genes involved in high-altitude adaptation in Tibetans. Genetic variants/haplotypes within regions containing three of these genes (EPAS1, EGLN1, and PPARA) are associated with relatively decreased hemoglobin levels observed in Tibetans at high altitude, providing corroborative evidence for genetic adaptation to this extreme environment. The mechanisms that afford adaptation to high-altitude hypoxia, however, remain unclear. Considering the strong metabolic demands imposed by hypoxia, we hypothesized that a shift in fuel preference to glucose oxidation and glycolysis at the expense of fatty acid oxidation would improve adaptation to decreased oxygen availability. Correlations between serum free fatty acid and lactate concentrations in Tibetan groups living at high altitude and putatively selected haplotypes provide insight into this hypothesis. An EPAS1 haplotype that exhibits a signal of positive selection is significantly associated with increased lactate concentration, the product of anaerobic glycolysis. Furthermore, the putatively advantageous PPARA haplotype is correlated with serum free fatty acid concentrations, suggesting a possible decrease in the activity of fatty acid oxidation. Although further studies are required to assess the molecular mechanisms underlying these patterns, these associations suggest that genetic adaptation to high altitude involves alteration in energy utilization pathways.

Energy power in mountains: difference in metabolism pattern results in different adaption traits in Tibetans / 中国应用生理学杂志

Energy metabolism plays an important role in life survival for species living in high altitude hypoxia condition. Air-breathing organisms require oxygen to create energy. Tibetans are the well-adapted highlanders in Qinghai-Tibetan Plateau. It was thought that different metabolic approaches could lead to different adaptation traits to high altitude hypoxia. Recently identified hypoxia inducible factors pathway regulators, endothelial PAS domain protein1 (EPAS1)/HIF-2a and PPARA, were involved in decreasing hemoglobin concentrations in Tibetans. Because EPAS1 and PPARA also modulated the energy metabolism during hypoxia, we hypothesized that positive selected EPAS1 and PPARA genes were also involved in unique energy metabolisms in Tibetans. In this brief review, we take a look into genetic determinations to energy metabolisms for hypoxia adaptations traits in Tibetans and mal-adaptive conditions such as high altitude diseases.

Genetic evidence for high-altitude adaptation in Tibet.

Tibetans have lived at very high altitudes for thousands of years, and they have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. These phenotypes are clearly the result of adaptation to this environment, but their genetic basis remains unknown. We report genome-wide scans that reveal positive selection in several regions that contain genes whose products are likely involved in high-altitude adaptation. Positively selected haplotypes of EGLN1 and PPARA were significantly associated with the decreased hemoglobin phenotype that is unique to this highland population. Identification of these genes provides support for previously hypothesized mechanisms of high-altitude adaptation and illuminates the complexity of hypoxia-response pathways in humans.

Changes of the c-Jun N-terminal kinase in the brains of rats with chronicfluorosis / 中国地方病学杂志

Objective To investigate the expression of c-Jun-N-terminal kinase(JNK) in rat brains with chronic fluorosis and try to reveal the molecular mechanism for the neural impairment induced by the disease.Methods The rats were randomly divided into 3 groups, normal control group(drinking water containing less than 0.5 mg/L of sodium fluoride, NaF), lower fluoride exposed group(drinking water containing 5 mg/L NaF) and higher fluoride exposed group(drinking water containing 50 mg/L NaF), 24 in every group. The rats were examined at the sixth month after feeding. The concentration of fluorine in urine and blood was detected by F-ion selective electrode. The expression of JNK in brains was investigated by using Western blotting and immunohitochemistry staining, and analyze the correlation between activating of JNK and the concentration of fluorine in blood. Results The increased concentration of fluorine in urine(control: 0.92 ± 0.30, lower fluoride exposed group: 2.56 ± 0.91,higher fluoride exposed group: 5.73 ± 3.14, P ＜ 0.05) were observed when 6 months after the beginning of the experiment, and the amount of fluorine in blood was also higher in rats with fluorosis(control: 0.12 ± 0.07, lower fluoride exposed group: 0.36 ± 0.14, higher fluoride exposed group: 0.50 ± 0.18, P ＜ 0.05). The expression of phospho-JNK at protein levels were higher in the brains of rats with fluorosis than that of controls (control: 1.00 ± 0.37, lower fluoride exposed group: 1.20 ± 0.28, higher fluoride exposed group: 1.74 ± 0.69, P ＜ 0.05), whereas no change of total-JNK was found(F = 0.046, P ＞ 0.05). Furthermore, the expression of phospho-JNK in the parietal cortex(119.3 ± 14.1), occipital cortex(112.7 ± 5.4), hippocampus CA3(100.6 ± 8.9), dorsal thalamus (117.8 ± 10.4) and olivary nucleus( 112.6 ± 5.9) of rats in higher fluoride exposed group were higher than that in control( 104.1 ± 8.9,106.6 ± 9.6,106.6 ± 9.7,108.9 ± 6.4,100.3 ± 8.4, all P ＜ 0.05) and lower fluoride exposed group(96.7 ± 17.1,102.5 ± 8.3,106.4 ± 6.5,110.2 ± 9.3,102.4 ± 4.7,102.5 ± 9.8, all P＜ 0.05). The positive stained neurons of total-JNK also distributed in the same brain regions of rats, but no difference was detected between the rats with fluorosis and controls(all P ＞ 0.05). The increased level of phospho-JNK was positively correlated with the fluoride contents in blood of the rats with fluorosis (r = 0.677). Conclusions The expression of phospho-JNK in brains of rats with fluorosis was significantly increased with a correlation to fluoride content in blood, which might be connected to the mechanism of neural impairment induced by chronic fluorosis.