iRhom2 Promotes Hepatic Steatosis by Activating MAP3K7-Dependent Pathway.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) has been widely recognized as a precursor to metabolic complications. Elevated inflammation levels are predictive of NAFLD-associated metabolic disorder. Inactive rhomboid-like protein 2 (iRhom2) is regarded as a key regulator in inflammation. However, the precise mechanisms by which iRhom2-regulated inflammation promotes NAFLD progression remain to be elucidated. APPROACH AND RESULTS: Here, we report that insulin resistance, hepatic steatosis, and specific macrophage inflammatory activation are significantly alleviated in iRhom2-deficient (knockout [KO]) mice, but aggravated in iRhom2 overexpressing mice. We further show that, mechanistically, in response to a high-fat diet (HFD), iRhom2 KO mice and mice with iRhom2 deficiency in myeloid cells only showed less severe hepatic steatosis and insulin resistance than controls. Inversely, transplantation of bone marrow cells from healthy mice to iRhom2 KO mice expedited the severity of insulin resistance and hepatic dyslipidemia. Of note, in response to HFD, hepatic iRhom2 binds to mitogen-activated protein kinase kinase kinase 7 (MAP3K7) to facilitate MAP3K7 phosphorylation and nuclear factor kappa B cascade activation, thereby promoting the activation of c-Jun N-terminal kinase/insulin receptor substrate 1 signaling, but disturbing AKT/glycogen synthase kinase 3ß-associated insulin signaling. The iRhom2/MAP3K7 axis is essential for iRhom2-regulated liver steatosis. CONCLUSIONS: iRhom2 may represent a therapeutic target for the treatment of HFD-induced hepatic steatosis and insulin resistance.

Nrf2 mitigates prolonged PM2.5 exposure-triggered liver inflammation by positively regulating SIKE activity: Protection by Juglanin.

Air pollution containing particulate matter (PM) less than 2.5 µm (PM2.5) plays an essential role in regulating hepatic disease. However, its molecular mechanism is not yet clear, lacking effective therapeutic strategies. In this study, we attempted to investigate the effects and mechanisms of PM2.5 exposure on hepatic injury by the in vitro and in vivo experiments. At first, we found that PM2.5 incubation led to a significant reduction of nuclear factor erythroid-derived 2-related factor 2 (Nrf2), along with markedly reduced expression of different anti-oxidants. Notably, suppressor of IKKε (SIKE), known as a negative regulator of the interferon pathway, was decreased in PM2.5-incubated cells, accompanied with increased activation of TANK-binding kinase 1 (TBK1) and nuclear factor-κB (NF-κB). The in vitro studies showed that Nrf2 positively regulated SIKE expression under the conditions with or without PM2.5. After PM2.5 treatment, Nrf2 knockdown further accelerated SIEK decrease and TBK1/NF-κB activation, and opposite results were observed in cells with Nrf2 over-expression. Subsequently, the gene loss- and gain-function analysis demonstrated that SIKE deficiency further aggravated inflammation and TBK1/NF-κB activation caused by PM2.5, which could be abrogated by SIKE over-expression. Importantly, SIKE-alleviated inflammation was mainly dependent on TBK1 activation. The in vivo studies confirmed that SIKE- and Nrf2-knockout mice showed significantly accelerated hepatic injury after long-term PM2.5 exposure through reducing inflammatory response and oxidative stress. Juglanin (Jug), mainly isolated from Polygonum aviculare, exhibits anti-inflammatory and anti-oxidant effects. We found that Jug could increase Nrf2 activation, and then up-regulated SIKE in cells and liver tissues, mitigating PM2.5-induced liver injury. Together, all these data demonstrated that Nrf2 might positively meditate SIKE to inhibit inflammatory and oxidative damage, ameliorating PM2.5-induced liver injury. Jug could be considered as an effective therapeutic strategy against this disease by improving Nrf2/SIKE signaling pathway.

Dysfunctional Rhbdf2 of proopiomelanocortin mitigates ambient particulate matter exposure-induced neurological injury and neuron loss by antagonizing oxidative stress and inflammatory reaction.

Ambient particulate matter (PM2.5)-induced metabolic syndromes is a critical contributor to the pathological processes of neurological diseases, but the underlying molecular mechanisms remain poorly understood. The rhomboid 5 homolog 2 (Rhbdf2), an essential regulator in the production of TNF-α, has recently been confirmed to exhibit a key role in regulating inflammation-associated diseases. Thus, we examined whether Rhbdf2 contributes to hypothalamic inflammation via NF-κB associated inflammation activation in long-term PM2.5-exposed mice. Specifically, proopiomelanocortin-specific Rhbdf2 deficiency (Rhbdf2Pomc) and corresponding littermates control mice were used for the current study. After 24 weeks of PM2.5 inhalation, systemic-metabolism disorder was confirmed in WT mice in terms of impaired glucose tolerance, increased insulin resistance, and high blood pressure. Markedly, PM2.5-treated Rhbdf2Pomc mice displayed a significantly opposite trend in these parameters compared with those of the controls group. We next confirmed hypothalamic injury accompanied by abnormal POMC neurons loss, as indicated by increased inflammatory cytokines, chemokines, and oxidative-stress levels and decreased antioxidant activity. These results were further supported by blood routine examination. In summary, our findings suggest that Rhbdf2 plays an important role in exacerbating PM2.5-stimulated POMC neurons loss associated hypothalamic injury, thus providing a possible target for blocking pathological development of air pollution-associated diseases.

Functional loss of inactive rhomboid-like protein 2 mitigates obesity by suppressing pro-inflammatory macrophage activation-triggered adipose inflammation.

OBJECTIVE: Chronic inflammation of adipose tissues contributes to obesity-triggered insulin resistance. Unfortunately, the potential molecular mechanisms regarding obesity-associated systemic inflammation and metabolic disorder remain complicated. Here, we report that inactive rhomboid-like protein 2 (iRhom2) was increased in overweight mice with adipose inflammation. METHODS: Mice with deletion of iRhom2 on a C57BL/6J background, mice without deletion of this gene (controls), and mice with deficiency of iRhom2 only in myeloid cells were fed a standard chow diet (SCD) or a high-fat diet (HFD; 60% fat calories). Then the adipose tissues or bone marrow cells were isolated for the further detection. RESULTS: After 16 weeks on a high-fat diet (HFD), obesity, chronic inflammation in adipose tissues, and insulin resistance were markedly mitigated in iRhom2 knockout (iRhom2 KO) mice, whereas these parameters were exaggerated in iRhom2 overactivated mice. The adverse influences of iRhom2 on adipose inflammation and associated pathologies were determined in db/db mice. We further demonstrated that, in response to an HFD, iRhom2 KO mice and mice with deletion only in the myeloid cells showed less severe adipose inflammation and insulin resistance than control groups. Conversely, transplantation of bone marrow cells from normal mice to iRhom2 KO mice unleashed severe systemic inflammation and metabolic dysfunction after HFD ingestion. CONCLUSION: We identified iRhom2 as a key regulator that promotes obesity-associated metabolic disorders. Loss of iRhom2 from macrophages in adipose tissues may indirectly restrain inflammation and insulin resistance via blocking crosslinks between macrophages and adipocytes. Hence, iRhom2 may be a therapeutic target for obesity-induced metabolic dysfunction.

Nrf2 deficiency aggravates PM2.5-induced cardiomyopathy by enhancing oxidative stress, fibrosis and inflammation via RIPK3-regulated mitochondrial disorder.

PM2.5 is a well-known air pollutant threatening public health, and long-term exposure to PM2.5 increases the risk of cardiovascular diseases. Nrf2 plays a pivotal role in the amelioration of PM2.5-induced lung injury. However, if Nrf2 is involved in PM2.5-induced heart injury, and the underlying molecular mechanisms have not been explored. In this study, wild type (Nrf2+/+) and Nrf2 knockout (Nrf2-/-) mice were exposed to PM2.5 for 6 months. After PM2.5 exposure, Nrf2-/- mice developed severe physiological changes, lung injury and cardiac dysfunction. In the PM2.5-exposed hearts, Nrf2 deficiency caused significant collagen accumulation through promoting the expression of fibrosis-associated signals. Additionally, Nrf2-/- mice exhibited greater oxidative stress in cardiac tissues after PM2.5 exposure. Furthermore, PM2.5-induced inflammation in heart samples were accelerated in Nrf2-/- mice through promoting inhibitor of α/nuclear factor κB (IκBα/NF-κB) signaling pathways. We also found that Nrf2-/- aggravated autophagy initiation and glucose metabolism disorder in hearts of mice with PM2.5 challenge. Cardiac receptor-interacting protein kinase 3 (RIPK3) expression triggered by PM2.5 was further enhanced in mice with the loss of Nrf2. Collectively, these results suggested that strategies for enhancing Nrf2 could be used to treat PM2.5-induced cardiovascular diseases.

Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK.

Nonalcoholic fatty liver disease (NAFLD) is featured by hepatic steatosis, insulin resistance, lipid deposition and inflammation. However, the pathogenic mechanism of NAFLD is still poorly understood. Dual-specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, has been reported to negatively modulate the mitogen-activated protein kinases (MAPKs) signaling, and it has never been investigated in NAFLD progression. In the study, we identified that DUSP16 could directly interact with TAK1 in human hepatocytes. DUSP16 knockdown in the isolated primary hepatocytes stimulated by palmitate (PA) showed accelerated lipid deposition and inflammatory response, along with the exacerbated activation of c-Jun NH2-terminal kinase (JNK), Transforming growth factor ß (TGF-ß)-activated kinase (TAK1) and nuclear factor-κB (NF-κB) signaling pathways; however, the opposite results were detected in PA-treated hepatocytes with DUSP16 over-expression. The in vivo experiments confirmed that DUSP16 knockout significantly aggravated the metabolic disorder and insulin resistance in high fat diet (HFD)-challenged mice. In addition, HFD-provoked hepatic lipid accumulation and inflammation were further promoted in mice with DUSP16 knockout through the same molecular mechanism as detected in vitro. Herein, these findings demonstrated that DUSP16 could directly interact with TAK1 and negatively regulate JNK signaling to alleviate metabolic stress-induced hepatic steatosis, and thus could be considered as a promising new molecular target for NAFLD treatment.

[Study on optimal harvest period of Lonicera Flos (Lonicera macranthoides)].

To ascertain the optimal harvest period of Lonicera Flos (Lonicera macranthoides) the configuration yield and quality of L. macranthiodes bloom verity and bud verity flower at different develop periods were Observed. The quality of L. macranthiodes which harvested at different times of the day was Compared. The configuration was significant difference between different develop period of L. macranthiodes flower. As bud growth, yield increased. Bloom verity of L. macranthoides chlorogenic acid content was significantly lower after opening (silver flower stage, golden flower stage), before opening (young bud stage, green-white stage) have no significant difference of the quality. Bud verity of L. macranthoides macranthoidin B is significant lower at yellow-white stage, young bud stage and green-white stage have no significant difference of the quality. The chlorogenic acid and isochlorogenic acid A content is significant difference between L. macranthoides harvested at different time of the day. The optimal harvest period of bloom verity is the white stage, picking time for 10:00 before and after 18:00. The optimal harvest period is the green-white stage, picking time is 8:00 before and after 18:00.

[Effect of fibrous root extract of Coptis chinensis on soil microbes and enzyme activities].

Coptis chinensis is widely used as Chinese medicine herbs and serious soil problems occur after continual cultivation of this medicinal plant. In the preset experiment, fibrous root extract of C. chinensis (REC) was added into soil to study the effect of REC on microbes and enzyme activity in soil. The results showed that both bacteria and actinomycetes decreased by about 2 times in contrast to fungi, which increased by about 3 folds. Phosphorus bacteria, potassium bacteria, azotobacter, ammonia bacteria, and nitrifying bacteria were also reduced significantly by REC, suggesting the inhibition of nitrogen biofixation and supply, mobilization of phosphorus and potassium, ad plant growth promotion as REC added into soil. There were multiple influences of REC on soil enzyme activities. Invertase activity was stimulated, while urease was inhibited and dehydrogenase unchanged by REC, indicating the interference of biochemical reactions in soil. In addition, type and total content of phosphorus lipid fatty acids (PLFAs) , the signature of microbes, decreased while the ratio of bacterium to fungus PLFAs increased as REC increased in soil, which suggested that fungi increased relatively with bacteria decreased thereby leading to easy occurrence of crop fungus diseases following cultivation of C. chinensis. The decrease in diversity and evenness indexes of microbial community in soil by REC indicated soil ecosystem deterioration and reduction of microbial groups and densities in soil. Therefore, allelopathic chemicals released from the roots of C. chinensis could change microbial community structure and resulted in serious soil problems by continual cropping of this medicinal plant.

[Effect of fertilization on phenolic components and antioxidant activities of Artemisia annua].

OBJECTIVE: A pot experiment with variable fertilizer treatments was carried out to study the influence of fertilization on the concentration and accumulation of polyphenols, scopoletin, chrysosplenol-D and chrysosplenetin in roots, stems and leaves and their antioxidant activities. The main aims were to fertilize scientifically in cultivation of Artemisia annua and improve the quality of the harvest organs. METHOD: These active components in leaves, stems and roots in the squaring stage were analyzed by HPLC and antioxidant activities of the extracts were evaluated by ultraviolet visible light colorimetric method. RESULT: The result showed the highest concentration of polyphenols, scopoletin, chrysosplenol-D and chrysosplenetin was in leaves, followed by stems and the lowest in roots. The antioxidant activities of the leaf extracts correlated positively with the concentrations of polyphenols, scopoletin, chrysosplenol-D and chrysosplenetin. Furthermore, fertilization promoted significantly the growth of A. annua, the biomass was increased by 57.37% (chemical fertilizer), 91.63% (mixture of chemical fertilizer and manure) and 92.27% (manure), respectively, compared to the blank control (without fertilizer). Fertilization, particularly mixture fertilization of chemical fertilizer and manure, increased generally the concentration and accumulation of polyphenols, scopoletin, chrysosplenol-D and chrysosplenetin as well as DPPH x scavenging ratio. CONCLUSION: Scopoletin, chrysosplenol-D and chrysosplenetin could be synthesized and stored mainly in leaves. The leaves might thus be the chief organ of A. annua for medical treatment. Finally, the mixture fertilization of chemical fertilizer and manure should be used to increase the yield and quality of A. annua.

[Allelopathic effect of artemisinin on green algae].

To study the growth effects of differing concentrations of artemisinin on green algae and to evaluate the ecological risk. The effects of artemisinin on the growth and the content change of chlorophyll, protein, oxygen, conductivity, SOD, CAT, MDA in Chlorella pyrenoidosa and Scenedesmus oblique were studied through 96 h toxicity tests. Artemisinin accelerated the growth of algae at a lower concentration ( <40 microg . L-1) with content increase of chlorophyll or protein and so on, and it inhibited the growth of algae at higher concentration ( >80 microg . L-1). The content of chlorophyll or protein in algae cells reduced with the increasing concentration of artemisinin, exhibiting the good concentration-effect relationship. SOD and CAT activity was stimulated at low concentrations ( <40 microg . L-1 ) and inhibited at high concentrations ( >80 microg . L- 1). However, MDA content increased significantly with the increase of concentration. According to the seven kinds of indicators changes, the time-response and dose-response suggested that the surfactant first hurt in Ch. pyrenoidosa was damaging membrane by changing membrane lipid molecules soluble. And primary mechanism on Chlorophyta cells might be related to the oxidation damage of lipid and other biological large molecules caused by artemisinin. The large-scale intensive planting of Artemisia annua may reduce the surrounding water productivity.

[Interspecific association of companion species in Artemisia annua community in southwest of China].

The interspecific association of companion species in Artemisia annua community in 48 region of southwest China was analyzed by variance analysis, chi2-test and association indices. The total related variance ratio among species in A. annua community was 2.05. Among 45 species pairs of 10 main species in the community, only 4 pairs showed significant negative correlations. Chi2 test, PC, OI, DI and AC values indicated pairs 1-8 (A. annua- A. lactiflora), 1-9 (A. annua- Setaria viridis) and 1-10 (A. annua- Bidens pilosa) showed a high correlations, and common utilization to non-restrictive resources. The results indicated that there was a significant positive correlation among species,and the community was at a stable stage, showed strong ability to human interference.

[Allelopathic effects of extracts from fibrous roots of Coptis chinensis on two leguminous species].

An experiment was carried out to study the allelopathic effects of Coptis chinensis fibrous root extracts (CRE) on the germination and seedling growth of Vicia faba and Pisum sativum in order to alleviate the allelopathic effects and increase land productivity. The seeds of both garden pea (P. sativum) and broad been (V. faba) were germinated in CRE solution of various concentrations, the germination rate, seedling growth and related physiological indexes were measured. The result indicated that there were no significant effects of CRE in low concentrations on seed germination, including both the rate and index, and seed vitality and membrane permeability. With the increment of CRE concentrations, however, the high seed membrane permeability and germination inhibition were observed. For example, the germination rates were reduced by 23.4% (P. sativum) and 9.5% (V. faba), respectively, in CRE solution with 800 mg . L-1. Simultaneously, soluble sugars and the free amino acids in the seeds were lower than those in the control (without CRE) after soaking seeds in CRE solutions. In addition, the seedling growth and nitrate reductase activity were stimulated by CRE at low concentrations in contrast to high concentrations which behaved otherwise and inhibited the nutrient utilization in endosperm. Therefore, the large amount of allelochemicals released from the roots and remains of C. chinensis in soils could inhibit the seed germination and seedling growth of legumes, which may lead to decrease even fail crop yields after growing this medical plant.

[Research on population dynamics of Lonicera macranthoides aphid and natural enemy in Xiushan and evolution of pesticides].

OBJECTIVE: To study the population dynamics of aphid on Lonicera macranthoides and their natural enemy in Xiushan and control method of pesticide so as to provide scientific basis for its integrated pests management (IPM). METHOD: The field investigation and the field controlling trial were carried out for the research. RESULT: Semiaphis heraclei was the dominant species among L. macranthoides aphids. The population dynamics of apterous aphids went through five consecutive stages: initial, fluctuating, rising, peak and declining. The population dynamics of alate aphids was 4-7 days later than that apterous aphid's. Significant positive correlations were found between the population size of spiders and ladybugs which were natural enemies and number of aphids. The result of pesticides against aphids in field trial showed that 25% thiamethoxam WG, 70% imidacloprid WG and 20% acetamiprid WP had well controlling effect. CONCLUSION: Aphids on L. macranthoides could be well controlled while 25% thiamethoxam WG, 70% imidacloprid WG and 20% acetamiprid WP are sprayed during the period of aphid population raising, the early April to the mid May.

[Studies on allelopathic effect of artemisinin on rhizobium].

Two strains of bean rhizobia, Rhizobium vigna 01 (slow-growing Rhizobium) and Rh. vigna 03 (fast-growing Rhizobium), were adopted to study allelopathic effect of artemisinin on the rhizobia. The results showed a significant inhibition of the reproduction and growth of rhizobium by artemisinin. After about 8 hours by adding 40 mg x L(-1) artemisinin into the culture medium, the number of rhizobia was less than half of those in normal culture. The utilization of sucrose and glucose by rhizobia decreased significantly as the concentration of artemisinin increased in the culture medium, which could be one of the main reasons for the inhibition of reproduction and growth of rhizobia by artemisinin. In addition, the activities of extracellular protease and acid phosphatase released from rhizobia decreased significantly as the concentrations of artemisinin increased. Artemisinin refluxed from Artemisia annua could thus inhibit the formation of root nodules and interfered with energy supply and reception between bacteroid and host cells. y = e(-ax) + b reflected the relationships between nitrogenase activities (y) and concentrations of artemisinin (x). In the culture medium with 48 mg x L(-1) of artemisinin, nitrogenase activities were about zero, resulting in the inactivation of nitrogenase in nodules formed. In general, artemisin in A. annua grown soils may inhibit the reproduction and growth of rhizobia, nodule formation and nitrogen biofixation, leading to less nitrogen supply, poor growth and development, and low yields of beans.

[Breeding and spreading of new vairety "Yu-Qing No. 1" of Artemisia annua].

OBJECTIVE: To breed and spread a new cultivar of Artemisia annua. METHOD: The excellent germplasm resources of A. annua in the main production area of Artemisia were collected, and the improved germplasm were screened, the content of artemisinin was determined, and yield per plant was measured. The systematically maternal line and seed production techniques of mass selection were applied combined with the variety test, variety regional test trials and production trials for breeding and spreading the new cultivars of artemisia. RESULT: The popularization and experiment illustrated the production of the new species reached 3 000 kg x hm(-2), compared with wild A. annua it increased 10% -14%. The content of artemisinin reached more than 1%, increased more than 0.2%. CONCLUSION: It is proved that the systematically maternal line and seed production techniques of mass selection can significantly improve the quality of A. annua and it is an acceptable way to cultivate new variety. By production verification, it is practicable and high technical and economic benefits to popularize the new cultivar "Yu-Qing No. 1" of A. annua.

[Development strategies on seed seedling of Chinese medicinal materials].

This paper analyzed the development status, industrialization system, standardization system and new variety breeding of seed and seedling of Chinese medicinal materials. Based on the development status and problems facing to the seed and seedling industry in China, some reasonable countermeasures are proposed in this article. The main measures include strengthening new variety breeding, establishing experiment site, protecting new variety and standardizing the market and quality management.

[Effects of zinc manganese and boron on artemisinin and yields of Artemisia annua].

OBJECTIVE: To explore the effects of micro-elements fertilizers on the quality and yields of Artemisia annua. METHOD: Field experiments were conducted according to the method of random blocks design. After the harvest the yield was calculated and the content of artemisinin was determined. RESULT: By applying 0.1%-0.5% Mn and 0.1%-0.5% Zn the dried leaf output and artemisinin content were increased. CONCLUSION: The suitable ranges of Mn and Zn can increased the yield and artemisinin content of A. annua.

[Optimal measure for cultivation of Artemisia annua with high seeds yield].

The relationship of Artemisia annua seed yield with density, N, P and K fertilizer applied amount was studied, and a mathematical model involving the 4 factors affecting seed yield was established using the orthogonal rotation design of quadratic regression. The seed yield function model was established according to parameters through field tests and data treated by computer techniques. The best agronomic measures complex project was selected and developed by computer imitation. The effects on seed yield of A. annua are density > N > P > K in turn. To obtain the highest yield density should be 13 000-15 000 plants x hm(-2), Ureal 186-242 kg x hm(-2), calcium superphosphate 874-1 023 kg x hm(-2), potassium chloride 135-165 kg x hm(-2) in the experiment. Reasonable planting density and fertilizer application could improve the seed yield of A. annua.

[Analysis of genetic difference among Scrophularia ningpoensis cultivars by SRAP].

OBJECTIVE: The genetic difference among Scrophularia ningpoensis cultivars were analyzed in molecular level. METHOD: Ninety-two individuls of three S. ningpoensis cultivars were employed to be analyzed by the approach of Sequence-related Amplified Polymorphism (SRAP).The parameters were calculated by POPGENE1.31 and the relationship was constructed based on UPGMA method. RESULT: 1) A total of 227 bands were scored and 199 bands of them were polymorphic. 2) The result is showed that there is a medium level of genetic diversity among three cultivars. At species level: percentage of polymorphic loci PPB=52.42%, effective number of alleles N(e)=1.2812, Nei's gene diversity H=0.1671 and Shannon's information index H(sp)= 0.2526; At cultivar level: PPB=21.44%, N(e)=1.1216, Nei's gene diversity H=0.0725 and Shannon's information index H(pop)= 0.1083. 3) The Nei's coefficient of genetic differentiation was 0.5625, which was consistent with the Shannon's coefficient of genetic differentiation (0.5713). Most of the genetic variation existed among cultivars. 4) The gene flow (N(m)=0.3889) was less among cultivars, indicating that the degree of genetic differentiation was higher. 5) Genetic similarity coefficient were changed from 0.8082 to 0.9133. By clustering analysis, the classified result of SRAP marker between traditional modal character was almost same. CONCLUSION: The genetic diversity of samples of S. ningpoensis is medium. The genetic difference among cultivar is higher than that within cultivar.