Effect of Dahuang Zhechong pills on long non-coding RNA growth arrest specific 5 in rat models of hepatic fibrosis

INTRODUCTION

Chronic liver diseases are still one of the most serious health problems in the world and hepatic fibrosis is the common outcome of these diseases.1,2Hepatic stellate cells(HSCs)are regarded as an important fibrogenic cell population in the liver,and activated HSCs promote hepatic fibrosis.3,4In addition,several inflammatory cytokines,including tumor necrosis factor alpha(TNF-α) and transforming growth factor beta(TGF-β),are recruited to the inflammatory site during the hepatic fibrosis process to aggravate the progression of hepatic fibrosis.5

Thirty male rats were maintained under the same conditions mentioned above,and randomly divided into three groups by random number table method,namely the CCl4-induced hepatic fibrosis model group,the CCl4plus DHZCP-treated group and the control group.In the first two groups,rats were injected intraperitoneally with a 0.6 mL/kg mixture of CCl4and corn oil(50%v/v corn oil and CCl4)twice per week for eight weeks.Rats in the control group were treated with equal volume corn oil.In addition,rats in the CCl4plus DHZCP group were fed with a 3.0 g/kg DHZCP(diluted with saline),every day during the eight weeks,while those in the other two groups were given equal volume of normal saline.Eventually,we euthanized the rats and collected their liver tissues for further research.

Due to the low efficiency of synthetic agents for treating liver diseases,including hepatic fibrosis,and the undesirable side effects,traditional Chinese medicine has been chosen as an alternative treatment for hepatic fibrosis.14Herbal medicines,such as the Fuzheng Huayu formula,have been proved to be a good choice for the treatment of hepatitis B-caused cirrhosis.15Another well-known and established Chinese herbal medicine,Dahuang Zhechong pill(DHZCP),can inhibit the proliferation of vascular smooth muscle cellsin vivoor hepatic fibrosis progression by suppressing the MAPK pathway.16,17However,the effects of lncRNAs in this process remain largely unknown.

In this study,we investigated the roles of the critical lncRNA GAS5 in a hepatic fibrosis rat model treated with DHZCP,and the interactive mechanisms of inhibiting hepatic fibrosis.

18 Zhang K,Gao Y,Zhong M,et al.Hepatoprotective effects of Dicliptera chinensis polysaccharides on dimethylnitrosamine-induced hepatic fibrosis rats and its underlying mechanism.J Ethnopharmacol 2016;179:38-44.

1．树立批判性思维教育理念，注重提高学生的思维能力。创新创业教育的改革应从树立批判性思维教育理念做起，应当倡导重视、提高学生的思维力，开发学生的思辨力，并将其纳入课程的主要目标。批判性思维教育理念认为，“教育与培训的使命:培养学生的批判性精神和独立思考的态度”，“批判性思维和创造性”是教育方式革新的重点，高等学校必须教育并培养学生，使其能够批判性地思考问题、分析问题。［8］这与创新创业教育的本质有相似之处。国外许多高校纷纷把培养大学生的批判性思维、促进大学生创新能力发展作为人才培养的重要目标和课程设置目标，批判性思维教育理念已经成为了整个教育界的核心理念之一，创新人才培养机制较为成熟。

MATERIALS AND METHODS

Drugs

DHZCP,purchased from Xi'an C.P.Pharmaceutical Co.,Ltd.,(Xi'an,China,lot No.61021089),contains Gancao(Radix Glycyrrhizae)90 g,Dahuang(Radix Et Rhizoma Rhei Palmati)300 g,Tubiechong(Eupolyphaga)30 g,Baishao(Radix Paeoniae Alba)120 g,Dihuang(Radix Rehmanniae)300 g,Qicao(Larva Holotrichiae)45 g,Taoren(Semen Persicae)120 g,Huangqi(Radix Astragali Mongolici)60 g,Mengchong(Tabanus)45 g,Shuizhi(Hirudo)60 g,Kuxingren(Semen Armeniacae Amarum)120 g and Gouqizi(Fructus Lycii)30 g.Hydrogen peroxide(30%,H2O2)was obtained from Guangzhou Wenliao Chemical Reagent Factory(China).

Preparation of DHZCP drug serum and normal serum

Male Sprague-Dawley rats,weighing 200-240 g,were randomly divided into drug serum group and normal serum group,and housed in a room at 20-25℃and relative humidity of 55%-60%.The research experimental procedures were approved by the Animal Care and Use Committee of Peking University Shenzhen Hospital.Rats in the control and DHZCP serum groups were treated by intragastrical administration of saline or 4.3 g/kg DHZCP for 10 d,respectively.The serum was serially prepared according to a previous study.16

Establishment of hepatic fibrosis rats model

Recent studies have shown that long non-coding RNAs(lncRNAs),more than 200 nucleotides in length,have diverse functions in various diseases,such as diabetes mellitus,liver cirrhosis and cancer.6-8Of particular interest in this study is the lncRNA growth arrest-specific 5(GAS5),which is considred as a type of tumor suppressor in various cancer.9-11In addition,overexpression of GAS5 can inhibit hepatic fibrogenesis by competing with endogenous RNA.12Thus,GAS5 may be associated with hepatic fibrosis and could be a target for potential therapy.12,13

3.2.6 出院指导 患者出院前,叮嘱患者要按时复查,一个月后来院拔除双J管,叮嘱患者要保持每天2L左右的饮水量,避免产生结石。饮食上不要过多的摄入奶制品,保持低动物蛋白和高纤维素的饮食原则。叮嘱患者置管阶段要进行自我护理,对血尿、尿频、尿急等等该症状进行观察,如果自觉不适,应该来源接受检查。

Cell line and cell culture

The rat HSC line,HSC-T6 cell line,was purchased from the Institute of Cell Research,Chinese Academic of Sciences(Shanghai,China).The HSC-T6 cells were cultured in DMEM(Dulbecco's Modified Eagle Medium,Gibco,Thermo Fisher,MA,USA)supplemented with 10%fetal bovine serum(Gibco,Thermo Fisher,MA,USA),100 ug/mL streptomycin and 100 U/mL penicillin,and incubated at 37℃with an atmosphere of 5%CO2.

Cell counting kit-8(CCK-8)assay

The cell proliferation assay was performed using CCK-8(Dojindo,Japan).Cells were seeded in 96-well plates supplemented with different concentrations of normal or drug serum from rats.Additionally,50 nM H2O2was used to promote cell proliferation for 72 h.Absorbance at a wavelength of 450 nm was measured by an ELISA microplate reader(Bio-Rad Laboratories Inc.,Hercules,CA,USA).Cells were also cultured in the presence of 15%serum,from normal or drug-treated rats,under the stimulation of 50 nM H2O2and absorbance was detected at the selected time points of 24,48 and 72 h.The experiments were repeated at least three times.

Immunohistochemistry staining

Liver tissue samples were collected from euthanized rats and were used for Masson staining and hematoxylin-eosin(HE)staining following fixation with 10%formalin according to the manufacturer's instructions.

Detection of activities of serum alanine aminotransferase(Alt),aspartate aminotransferase(Ast),Tnf-αand Tgf-β

Commercially available kits(Jiancheng Institute of Biotechnology,Nanjing,China)were used to detect the activities of Alt and Ast according to the manufacturer's protocol.The levels of cytokines Tnf-α and Tgf-β were measured using the rat cytokine multiplex kit(Linco Research Inc.,Saint Charles,MO,USA).The experiments were performed in triplicate.

RNA extraction and quantitative real-time polymerase chain reaction(qRT-PCR)analysis

Total RNA was extracted from tissues or cells using the TRIzol reagent(Invitrogen,Carlsbad,CA,USA)following the manufacturer's instructions.Total RNA was reverse transcribed to cDNA using the Prime Script RT Reagent Kit with gDNA Eraser(Takara,Dalian,China).The housekeeping gene Gapdh was chosen as an internal control.The qRT-PCR amplification reaction was performed as follows:5 min at 95℃;then 40 cycles of denaturation at 95℃for 15 s,annealing at 56℃ for 30 s,and extension at 72℃ for 30 s.The primers used were the following:GAS5 forward primer 5'-ATGGCAAGCTCCACACAAG-3',reverse primer 5'-TTCAAGCATCCATCCAGTCA-3',Gapdhforward primer 5'-CACGGCAAGT TCAACGGCACAGT-3',reverse primer 5'-AGCGGAAGGGGCGGAGATGAT-3',qRT-PCR analysis was performed on an ABI PRISM 7500 Fluorescent Quantitative PCR System(Applied Biosystems,Foster City,CA,USA)in triplicate according to the manufacturer's instructions.The comparative 2ΔΔCtmethod was used to calculate the relative amount of GAS5.

Overexpression of GAS5 in HSC-T6 cells

The GAS5 sequence was subcloned into the adenovirus shuttle plasmid vector pAd-EF1a-GFP(Vigene Biosciences,Jinan,China).Homologous recombination was used with the pBHGlox_E1,3 Cre and shuttle plasmid to generateAd-GAS5.Then,weused Ad-GAS5 to infect cells.The non-sense sequence,which was the same length as GAS5,was inserted into the same vector to be used as the negative control.

Western blot analysis

In conclusion,our findings suggest that DHZCP may alleviate hepatic fibrosis by increasing the expression of GAS5 to suppress p-Erk and by regulating other factors to inhibit p-p38.This may represent the interactive mechanism between GAS5 and DHZCP in hepatic fibrosis.

Statistical analysis

Data are expressed as mean±standard deviation(xˉ±s).The SPSS 19.0(IBM SPSS Statistics for Windows,Version 19.0.Armonk,NY,USA)was used to analyze all statistical data.The CCK-8 assay data were analyzed by analysis of variance and the two-tailedt-test was used and statistical significant level wasP＜0.05.

RESULTS

Effects of the serum of saline-treated or DHZCP-treated rats on H2O2-stimulated proliferation of HSCs

The results shown in Figure 1A reveal that,without the stimulation of 50 nM H2O2,the effect of varying concentrations of serum from DHZCP-treated rats on the proliferation of HSCs at 72 h shows no significant difference when compared with varying concentrations of serum from the normal serum group.On the other hand,with the stimulation of 50 nM H2O2,compared with the control group,cell proliferation was significantly inhibited in the DHZCP-treated group at a 15%or 20%concentration of serum(P＜0.01).However,there was no significant difference between the two groups at a 5%or 10%concentration of serum.Moreover,compared with the normal serum group,cell growth was arrested under the stimulation of H2O2at 15%concentration of serum in the DHZCP serum group(Figure 1B,P＜0.01).

Role of anti-hepatic fibrosis by DHZCP

In order to determine whether the hepatic fibrosis model had been successfully established,we used qualitative histological analysis with hematoxylin-eosin(HE)and masson staining.The results shown in Figure 2 indicate that compared with the control group(Figure 2A,D),collagen accumulation,fiber extension,fatty degen-eration and pseudolobule formation obviously occurred in the CCl4-induced hepatic fibrosis models(Figure 2B,E).However,the CCl4-induced hepatic fibrosis(Figure 2C,F)was significantly attenuated in the CCl4plus DHZCP group.

Figure 1 Proliferation of HSCs was detected by the CCK-8 assay

Figure 2 Representative images of pathological changes of liver in normal or hepatic fibrosis rats,HE and masson staining(×100)

A,D:normal liver tissues;B,E:CCl4induced fibrotic liver tissue;C,F:CCl4induced plus administration of DHZCP.A,B,C:HE staining;D,E,F:masson staining.HE:hematoxylin-eosin;DHZCP:Dahuangzhechong pill.

Inhibition of Alt and Ast serum activities by DHZCP

Compared with the control group,the activity of Alt was significantly increased in the CCl4group(Figure 3A).Meanwhile,no significant difference was detected in the activity of Ast between these two groups(Figure 3B).In addition,the results showed that DHZCP could significantly decrease activities of Alt and Ast compared with the CCl4group(P＜0.05).

Inhibition of serum cytokines of Tnf-αand Tgf-β

The changes of the serum cytokines Tnf-α and Tgf-β are shown in Figure 4.Both,the expression of Tnf-α and Tgf-β were substantially increased by the serum from the CCl4-treated rats at the end of experiment.Compared with the CCl4group,the DHZCP treatment markedly downregulated the expression of serum Tnf-α and Tgf-β.

Expression of GAS5 and MAPK phosphorylation

Figure 3 Serum expressions of Alt and Ast

A:Alt;B:Ast.Control group was administrated with normal saline.CCl4group was treated with CCl4intraperitoneally.DHZCP group was treated with CCl4intraperitoneally plus intragastrical administration of DHZCP.DHZCP:Dahuangzhechong pill.aP＜0.05,compared with control group andbP＜0.05,compared with CCl4group.

To investigate the roles of GAS5 and the MAPK pathway in hepatic fibrosis,we performed qRT-PCR and Western blot analysis to detect their relative expression.The results shown in Figure 5A reveal that,compared with the control group,the relative expression of GAS5 was significantly decreased in the CCl4group(P＜0.01).In contrast,DHZCP significantly increased the expression of GAS5 in the CCl4plus DHZCP group compared with the CCl4group(P＜0.05).In addition,compared with the control group,phosphorylation of p38(p-p38)and Erk(p-Erk)was markedly increased(P＜0.05),while no changes in the phosphorylation of Jnk(p-Jnk)was observed in the CCl4group(Figure 5B).Additionally,the levels of p-p38 and p-Erk in the CCl4plus DHZCP group were evidently reduced compared with the CCl4group(BothP＜0.05).

Overexpression of GAS5 and its effects on HSCs proliferation

Figure 4 Effects of DHZCP on serum expression of Tnf-α and Tgf-β in CCl4treated rats

A:the serum expression of Tnf-α;B:the serum expression of Tgf-β.Control group was administrated with normal saline.CCl4group was treated with CCl4intraperitoneally.DHZCP group was treated with CCl4intraperitoneally plus intragastrical administration of DHZCP.DHZCP:Dahuangzhechong pill.aP＜0.01,compared with control group;bP＜0.05,compared with CCl4group.

Figure 5 Expression level of GAS5 and Mapk of liver tissues in different groups

A:the expression levels of GAS5 in rats liver tissues;B:the protein expression levels of p38,Erk,Jnk and their phosphorylation in liver tissues.Actin was applied as control protein.1:Control group was administrated with normal saline.2:CCl4group was treated with CCl4intraperitoneally.3:DHZCP group was treated with CCl4intraperitoneally plus intragastrical administration of DHZCP.GAS5:growth arrest specific 5;DHZCP:Dahuangzhechong pill.aP＜0.01,compared with control group;bP＜0.05,compared with CCl4group.

Figure 6 Effects of the over expression of GAS5 on the proliferation and Mapk phosphorylation in HSCs

A:schematic diagram of the adenovirus shuttle plasmid vector pAd-EF1a-GFP.B:the expressions of GAS5 in untreated cells,and cells infected with Ad-vector and Ad-GAS5 after 72 h.C:the OD450(optical density)value of cell groups.D:the protein expression levels of p38,Erk,Jnk and their phosphorylation in cell groups.Actin was applied as control protein.Under the stimulation of 50 nM H2O2,Hepatic stellate cells were treated with 1(normal serum plus Ad-vector),2(drug serum plus Ad-vector),3(normal serum plus Ad-GAS5)and 4(drug serum plus Ad-GAS5).aP＜0.01,compared with Ad-vector group;bP＜0.05,compared with normal serum plus Ad-vector group;andcP＜0.05,normal serum plus Ad-GAS5 group.GAS5:growth arrest specific 5;HSCs:hepatic stellate cells.

The schematic diagram of the adenovirus shuttle plasmid vector pAd-EF1a-GFP is presented in Figure 6A.The untreated group represented the HSCs that were not infected with adenovirus.Compared with the negative control group and the untreated group,the expression of GAS5 was significantly increased in cells transfected with Ad-GAS5(Figure 6B,bothP＜0.01).Cells were stimulated with H2O2in all testedconditions and,as shown in Figure 6C,without overexpression of GAS5,the proliferation of HSCs cultured with serum from DHZCP-treated rats was significantly suppressed at the 72 h time point compared with cells cultured with normal serum(P＜0.05).In the normal serum group,after overexpression of GAS5,cell growth was significantly inhibited(Figure 6C,P＜0.05).Likewise,the growth of cells that were cultivated with serum from DHZCP-treated rats was markedly arrested when cells overexpressed GAS5(P＜0.05).In addition,we found that p-Erk was significantly inhibited when cells were cultured with the DHZCP drug serum or overexpressed GAS5 compared with the control group.The efficiency of the p-Erk inhibition is increased when the drug serum and GAS5 overexpression conditions are combined(Figure 6D).Furthermore,p-p38 was suppressed when the cells were cultured in drug serum,while overexpression of GAS5 had no obvious effect on the level of p-p38.Together,the results suggest that DHZCP can inhibit hepatic fibrosis through regulation of the p38 and Erk pathway and DHZCP may suppress the Erk pathway through regulation of the expression level of GAS5.

DISCUSSION

Traditional Chinese medicine remedies have been used as complementary drugs to treat hepatic diseases.18-20The advantages of traditional Chinese medicine treatment for liver disease include:protecting hepatocytes,scavenging oxygen free radicals,improving hepatic microcirculation, and inhibiting hepatic fibrosis.21DHZCP has herbal components that possessing antihepatic-fibrosis activity.Scutellariae radix and paeoniae radix alba can reduce the expression of α-SMA,as well as the levels of serum hyaluronic acid,liver hydroxyproline and malondialdehyde induced by CCl4.22,23Other components of DHZCP have also been found to exert an anti-fibrosis effect by promoting apoptosisviacaspase 3/9 activation,inhibiting HSCs activation and proliferation,attenuating the accumulation of ECM and improving the function of the liver.24-26In this study,we found that DHZCP significantly reduced collagen accumulation and hepatic fibrosis.Moreover,the activities of Alt,Ast,Tnf and Tgf-β were suppressed in the CCl4plus DHZCP group compared with the CCl4group.These results are consistent with similar findings in a previous study.17

我国刑法第285条第二款非法获取计算机数据罪、非法控制计算机系统罪规定：“违反国家规定，侵入前款规定以外的计算机信息系统或者采用其他技术手段，获取该计算机信息系统中存储、处理或者传输的数据，或者对该计算机信息系统实施非法控制，情节严重的，处三年以下有期徒刑或者拘役，并处或者单处罚金；情节特别严重的，处三年以上七年以下有期徒刑，并处罚金。”

黄名选 男，1966年出生于广西乐业县，工学硕士，现为广西财经学院计算机系教授，主要研究方向为数据挖掘、信息检索、机器学习，主持国家自然科学基金项目2项，主持完成广西自然科学基金项目1项，主持广西教育厅科研项目3项，获2011年广西高校优秀人才资助计划项目1项，参与完成国家自然科学基金项目1项，发表学术论文60 余篇，其中，中文核心期刊论文40余篇，被期刊EI 收录4篇，ISTP 收录1 篇，授权的发明专利9 件.

HSCs are found in a quiescent state in the healthy liver.However,HSCs can change into an activated state in response to liver damage.Continued activation of HSCs is an essential part in the process of hepatic fibrosis.On the one hand,activated HSCs can promote cell proliferation and be involved in the reconstruction of the liver structure by secreting excessive ECM.On the other hand,activated HSCs can also elevate the liver sinus internal pressure by cell shrinkage.These two types of change eventually lead to hepatic fibrosis and portal hypertension.In the present study,HSCs were activated with 50 nM H2O2.After exposure to the serum of rats that have been treated with DHZCP,the proliferation of HSCs was significantly decreased and exhibited a very clear dose-and time-effect relationship with DHZCP.

In recent years,several studies about lncRNAs have reported their involvement in numerous biological processes of diseases.27LncRNAs may also play vital roles in hepatic fibrosis.For instance,the lncRNA HIF1AAS1 interacts with TET3 and participates in the activation of HSCs.28Knockdown of the lncRNA APTR inhibits the activation of HSCs by regulating p21.29Additionally,the expression level of the lncRNA H19 can regulate HSCs proliferation through targeting IGF1R.30Accordingly,due to the significant inhibitory role of the lncRNA GAS5 in HSCsin vitroandin vivo,12we hypothesized that it may represent an important potential target for hepatic fibrosis therapy and needs to be further studied.During hepatic fibrosis,HSCs are responsible for synthesis of collagen and in this study,results show that the proliferation of HSCs was significantly inhibited after overexpression of GAS5.In addition,in this research,we investigated their mutual relationship and found a more effective potential method to treat hepatic fibrosis.Specifically,the results demonstrated that compared with the group treated with CCl4only,DHZCP can reverse the relative mRNA expression of GAS5 in hepatic tissue samples from rats treated with CCl4plus DHZCP.

It has been reported that GAS5 is involved in cancer suppressionviathe regulation of EGFR and its downstream signaling proteins,including p-AKT,and p-ERK.31MAPKs,including p38,ERK,and JNK,play an important role in the activation of HSCs and in anti-hepatic fibrosis.DHZCP can exert an anti-hepatic fibrosis effect by downregulating the expression of phosphorylated Erk and p38,but not Jnk.17The results of the current study suggest that DHZCP can significantly promote the expression of GAS5 and suppress p38 and Erk phosphorylation.We also used an adenovirus transfection method to overexpress GAS5.Our results indicated that GAS5 overexpression,significantly suppressed Erk phosphorylation but not that of p38.Accordingly,this indicates that GAS5 may attenuate hepatic fibrosis by downregulating Erk but not p38 phosphorylation.

Liver tissue samples and cells were lysed with the Protea JET Mammalian Cell Lysis Reagent(Fermentas inc.,Burlington,Ontario,Canada)containing phosphatase inhibitor cocktail(Pierce Biotechnology Inc.,Rockford,IL,USA)and a cocktail of proteinase inhibitors(Roche,Penzberg,Germany).Total protein was quantified using a BCA protein assay kit(Boster,Wuhan China).The protein was run and separated on 10%SDS-PAGE and transferred onto PVDF membranes(Millipore,Billerica,MA,USA).After blocking,the membranes were probed with rabbit monoclonal antibodies against rat SAPK/JNK(56G8)(CST,USA),p44/42 MAP Kinase(137F5)(CST,USA),phospho-p44/42 MAPK(Thr202/Tyr204)(CST,USA),phosphop38 MAPK(Thr180/Tyr182)(CST,USA),p38 MAP kinase(CST,USA),phosphor-SAPK/JNK(Thr183/Tyr185)(CST,USA),and mouse monoclonal antibody against rat-actin(Santa Cruz Biotechnology,Santa Cruz,CA,USA)as primary antibodies at 1∶1000 dilution for 1 h.Goat anti-mouse IgG labeled with HRP(1∶1000)was used as the secondary antibody.ECL-chemiluinescent kit(ECL-plus,Thermo Scientific,Waltham,MA,USA)was used to detect the immunoreactive signals of the protein.Films were scanned,and analyzed by densitometry using Image J 1.43 software,to quantify the signal intensity of the protein bands.

REFERENCES

CONCLUSIONS:DHZCP can alleviate hepatic fibrosis by increasing the expression of GAS5 to suppress p-Erk and regulating other factors to inhibit p-p38.

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解析：设调整比例前后A同化的能量均为M，调整前后C获得的能量分别为E1、E2，则E1=M×10%×1/2+M×10%×1/2×10%=11M/200；E2=M×10%×4/5+M×10% ×1/5×10%=41M/500。 E2/E1=41M/500÷（11M/200）=82/55≈1.49。故调整比例后该生态系统能承载C的数量是原来的1.49倍。

1、水域监测。监测结果表明，2012年库区的河流、水库、坑塘这三类的水域面积共173309.96亩，2017年水域面积192135.43亩，五年间增加水域面积18826.47亩，其中水库面积增加17850.55，占94.81%，主要为涔天河水库扩建后蓄水所致。据统计，水库淹没区域面积共14720.12亩，其中林地面积最多，达8136.86亩，耕地面积其次，为2574.71亩。对水库进行淹没模拟分析表明，当涔天河水库水位达正常蓄水位313米时，模拟淹没情况显示水库面积达44076亩。当水位达设计洪水位317.76 m时，模拟淹没情况显示水库面积达47305亩。

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注意：有消化不良时，消食药最多吃7天，如症状没改善应及时就诊。口感好的消食药一般都加了蜂蜜、蔗糖，糖友慎用。经常出现嗳气、胃疼，应到医院排除其他病，遵医嘱治疗。

10 Sun M,Jin FY,Xia R,et al.Decreased expression of long noncoding RNA GAS5 indicates a poor prognosis and promotes cell proliferation in gastric cancer.BMC Cancer 2014;14:319.

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经历了连续超过两周的低迷行情后，尿素价格终于止跌维稳。这一波行情触底，还要归功于国际方面。11月19日，印度中标180万吨，其中中国货源中标93万吨，这无疑为国内行情注入了一剂强心剂。受此影响，山东、河北等地尿素厂家出口新单明显增多，11月20日各地尿素企业预收款情况也明显好转，尿素价格随着新单跟进和厂家有意挺价之下也基本持稳。考虑当前已进入11月下旬，未来短期内尿素市场需求将有所回暖，价格也再难有跌幅。

19 XiS,ShiM,Jiang X,et al.The effects of Tao-Hong-Si-Wu on hepatic necroinflammatory activity and fibrosis in a murine model of chronic liver disease.J Ethnopharmacol 2016;180:28-36.

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21 Zhao CQ,Zhou Y,Ping J,Xu LM.Traditional Chinese medicine for treatment of liver diseases:progress,challenges and opportunities.J Integr Med 2014;5(12):401-408.

22 Nan JX,Park EJ,Kim YC,Ko G,Sohn DH.Scutellaria baicalensis inhibits liver fibrosis induced by bile duct ligation or carbon tetrachloride in rats.J Pharm Pharmacol 2002;4(54):555-563.

同时，CS渠道对于大宝的高端化也表示期待。对于以后是否会引进大宝的这款产品，百强美妆连锁负责人表示会继续观察，持续保持关注。

23 Qin Y,Tian YP.Protective effects of total glucosides of paeony and the underlying mechanisms in carbon tetrachloride-induced experimental liver injury.Arch Med Sci 2011;4(7):604-612.

24 Park EJ,Zhao YZ,Lian L,Kim YC,Sohn DH.Skullcapflavone I from Scutellaria baicalensis induces apoptosis in activated rat hepatic stellate cells.Planta Med 2005;9(71):885-887.

25 Han YL,Li ZW.Purification and activity calculated of fibrinolyric factors from Eupolyphaga sinensis.Zhong Yao Cai 2006;8(29):765-767.

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首先从犯罪主体的年龄上来说，必须是年满十六周岁的具有刑事责任能力的自然人，同时这里的醉酒需要达到一定的标准，如果不符合标准只能认定为“酒后驾驶”，达不到醉驾标准只需要承担相应的行政责任，而不需要承担刑事责任。其次，有些学者在该罪名主体的研究上，会将无证驾驶和醉酒驾驶进行某种程度上的混淆，因为我国新的道路交通法规定，无证驾驶的行为人需要处以一定的行政拘留和罚款，但是无证同时醉驾的行为人是不是构成醉酒型危险驾驶罪呢？笔者认为这个答案是肯定的。醉驾之所以受到处罚是出于对其行为的危害性考虑，跟其行为人有没有驾驶证并没有直接的影响，可以作为醉驾行为的一个严重结果。

27 Zhao XY,Lin JD.Long noncoding RNAs:a new regulatory code in metabolic control.Trends Biochem Sci 2015;10(40):586-596.

28 Zhang QQ,Xu MY,Qu Y,et al.TET3 mediates the activation of human hepatic stellate cellsviamodulating the expression of long non-coding RNA HIF1A-AS1.Int J Clin Exp Pathol 2014;11(7):7744-7751.

29 Yu F,Zheng J,Mao Y,et al.Long non-coding RNA APTR promotes the activation of hepatic stellate cells and the progression of liver fibrosis.Biochem Biophys Res Commun 2015;4(463):679-685.

30 Yang JJ,Liu LP,Tao H,et al.MeCP2 silencing of LncRNA H19 controls hepatic stellate cell proliferation by targeting IGF1R.Toxicology 2016;(359-360):39-46.

31 Dong S,Qu X,Li W,et al.The long non-coding RNA,GAS5,enhances gefitinib-induced cell death in innate EGFR tyrosine kinase inhibitor-resistant lung adenocarcinoma cells with wide-type EGFRviadownregulation of the IGF-1R expression.J Hematol Oncol 2015;8:43.