• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Hua-Zhuo-Kai-Yu decoction inhibits apoptosis in nonalcoholic fatty liver disease

    2021-12-04 06:54:46YuTingLiHuanTianCuiLuYangLuLuJinYuMingWangXueQianDongWeiBoWenHongWuWangZhaiYiZhang
    Traditional Medicine Research 2021年1期

    Yu-Ting Li, Huan-Tian Cui, Lu Yang, Lu-Lu Jin, Yu-Ming Wang, Xue-Qian Dong, Wei-Bo Wen, Hong-Wu Wang,Zhai-Yi Zhang

    1College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China;2Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao 250100, China; 3Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 301617,China; 4Department of Endocrinology in Yunnan Provincial Hospital of Traditional Chinese Medicine, Kunming 650021,China; 5College of Integrated Chinese and Western Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617,China.

    Abstract

    Background:Hua-Zhuo-Kai-Yu decoction(HZKY)is an empirical formula in traditional Chinese medicine that is derived from the classic ancient prescription Da-Chai-Hu decoction.It has been demonstrated to have good clinical effects on nonalcoholic fatty liver disease (NAFLD).However, the mechanism by which HZKY acts on NAFLD remains unclear.In this study,network pharmacology was used to predict the potential targets of HZKY in NAFLD.Additionally, in vivo studies were conducted to validate the crucial pathways determined using network pharmacology.Methods: Active compounds in HZKY were screened using the Traditional Chinese Medicine Systems Pharmacology and Analysis Platform and Traditional Chinese Medicine Integrated Database, and the potential targets of compounds in HZKY were predicted using Traditional Chinese Medicine Systems Pharmacology and Analysis Platform, Traditional Chinese Medicine Integrated Database, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and PUBCHEM.In addition,targets involved in NAFLD were obtained from the GeneCards and Online Mendelian Inheritance in Man databases,and the potential targets of HZKY in NAFLD were identified based on the common potential targets between HZKY and NAFLD.Cytoscape 3.7.2 was used to visualize crosstalk and identify the key genes from the potential targets of HZKY in NAFLD.Kyoto Encyclopedia of Genes and Genomes analysis was conducted to predict the pathways by which HZKY acts on NAFLD.Rats were fed with a high-fat diet for 12 weeks to induce NAFLD and were then orally administered HZKY.Serum lipid levels and hematoxylin and eosin and oil red O staining results were assessed to determine the effects of HZKY in NALFD.Furthermore, the mechanisms of action of HZKY in NAFLD, as determined using network pharmacology, were validated based on the inhibition of apoptosis in the liver using Western blotting.Results:A total of 269 potential targets of 130 active compounds in HZKY were identified(oral bioavailability ≥30% and drug-like ≥0.18), and 62 targets were selected after being compared with the targets of NAFLD.Bcl-2-associated X protein (BAX), caspase3 (CASP3), and caspase9 (CASP9) were the key genes with the highest values of network connectivity.In addition, 45 Kyoto Encyclopedia of Genes and Genomes pathways,including apoptosis, fatty acid synthesis, and estrogen signaling, were enriched according to the selected genes of HZKY.In vivo studies showed that the serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol were significantly elevated and the serum level of high-density lipoprotein cholesterol was decreased in the model group compared with those in the control group(P <0.01 for all).The expressions of BAX,CASP9,and CASP3 were upregulated in the model group compared with those in the control group(P <0.05,P <0.01,and P <0.01, respectively), while HZKY treatment decreased the body weights and serum levels of total cholesterol,triglyceride, and low-density lipoprotein cholesterol and increased the serum levels of high-density lipoprotein cholesterol in NAFLD model rats(P <0.05,P <0.01,P <0.05,and P <0.05,respectively).Hematoxylin and eosin and oil red O staining indicated that HZKY treatment reduced steatosis in the hepatocytes.Moreover, HZKY treatment inhibited apoptosis in the liver by downregulating the expressions of BAX, CASP3, and CASP9 (P <0.05, P <0.01,and P <0.05,respectively).Conclusion:Our study demonstrates that HZKY improves NAFLD by inhibiting apoptosis in the liver by reducing the levels of BAX,CASP3,and CASP9.

    Key words:Network pharmacology,Hua-Zhuo-Kai-Yu decoction,Nonalcoholic fatty liver disease,Apoptosis

    Backgro und

    Nonalcoholic fatty liver disease(NAFLD)is caused by metabolic liver injury accompanied by extensive hepatic steatosis.Epidemiological studies have indicated that the morbidity of NAFLD is approximately 6.3%-45% in adults and 15% in developed areas such as Beijing, Shanghai, and Guangzhou in China [1, 2].Long-term NAFLD can cause chronic metabolic diseases such as diabetes,liver fibrosis, liver cirrhosis, and hepatocellular carcinoma [3, 4].Currently, clinical treatment for NAFLD includes administration of insulin sensitizers,antioxidants, and lipid-lowering drugs.However, these drugs have been shown to have multiple side effects.Lipid-lowering drugs can trigger the dysfunction of hepatic enzymes [5].Insulin sensitizers and antioxidants have been shown to induce water-sodium retention and increase the risk of cardiac diseases[6].

    Traditional Chinese medicine (TCM) has been demonstrated to have better treatment effects on NAFLD.Ling-Gui-Zhu-Gan decoction, which is a classic ancient TCM prescription, can attenuate high-fat diet (HFD)-induced NAFLD by improving insulin resistance (IR) and regulating lipid metabolism-related pathways [7].The empirical formula of the TCM Huo-Xue-Xiao-Zhi decoction can decrease tumor necrosis factor-α, transforming growth factor, and free fatty acid levels and improve hepatic function in patients with NAFLD [8].Additionally, the empirical formula of the TCM Yi-Qi-Huo-Xue-Qu-Tan can modulate the dysfunction of lipid metabolism in these patients[9].

    The classic ancient prescription Da-Chai-Hu (DCH)decoction (Chaihu (Bupleuri Radix), Huangqin(Scutellariae Radix), Shaoyao (Paeoniae Radix Alba),Banxia (Pinelliae Rhizoma), Shengjiang (Zingiberis Rhizoma Recens),Zhishi(Aurantii Fructus Immaturus),Dazao (Jujubae Fructus)) is described in the ancient book on Chinese medicine titledShanghan Zabing Lun(Treatise on Cold Damage Diseases,25 C.E.-220 C.E.), written by Zhang Zhongjing.According to the book,DCH is used to treat diseases of the liver,gallbladder, and pancreas.Recent studies have also demonstrated the hepatoprotective, cholagogue,anti-inflammatory, and lipid-lowering effects of DCH[10, 11].In addition, clinical studies have shown that DCH improves dyslipidemia in patients with NAFLD[12].Hua-Zhuo-Kai-Yu (HZKY) decoction, derived from a modification of DCH, was established by Doctor Wang Hongwu at the Tianjin University of Traditional Chinese Medicine.It has shown obvious clinical effects on NAFLD.However, the mechanisms of action of HZKY in NAFLD remain unknown.

    Network pharmacology, developed by Hopkins AL in England, is the study of the relationships among drugs, diseases, genes, and targets based on the combination of subject theory, including systematic biology, pharmacology, and multiple “omics” studies such as genomics and proteomics [13-16].The application of network pharmacology in studying the mechanisms of TCM is in line with the characteristics of TCM, including a systematic and holistic perspective with multiple components.It is developing into an effective tool for identifying the overall mechanisms of TCM drugs[17].In this study,network pharmacology was used to predict the potential targets of HZKY in NAFLD.Furthermore, rats were fed with HFD for 12 weeks to induce NAFLD and were then administered HZKY orally.Serum lipid levels and hematoxylin and eosin (H&E) and oil red O staining results were assessed to observe the effects of HZKY in NALFD.Moreover, the mechanisms of action of HZKY in NAFLD, as determined using network pharmacology, were validated using Western blotting,which showed apoptosis inhibition in the liver.

    Materials and Methods

    Network Pharmacology Study

    Identification of the main active compounds.HZKY is composed of Chaihu 12 g (Bupleuri Radix),Huangqin 9 g (Scutellariae Radix), Shaoyao 9 g(Paeoniae Radix Rubra), Zhishi 9 g (Aurantii Fructus Immaturus), Banxia 9 g (Pinelliae Rhizoma), Dahuang 6 g (Rhei Radix et Rhizoma), Bohe 9 g (Menthae Haplocalycis Herba), Heye 9 g (Nelumbinis Folium),and Peilan 9 g (Eupatorii Herba).The main compounds in HZKY were screened using the Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP)(http://tcmspw.com/tcmsp.php) and Traditional Chinese Medicine Integrated Database (TCMID)(http://www.megabionet.org/tcmid).The repeated compounds were counted only once.

    Because of the oral administration of HZKY for the treatment of NAFLD, oral bioavailability (OB) was regarded as the pharmacokinetic parameter for HZKY to be considered in the network pharmacology evaluations.The compounds in HZKY (OB ≥30%),which were considered to be absorbed, metabolized,and functioning in the human body,were chosen as the candidate compounds [18].Then, the structural and physiochemical properties of candidate compounds in HZKY were identified using the DrugBank(https://www.drugbank.ca/) database.Compounds that were drug-like (DL) ≥0.18 were identified as the main active compounds of HZKY[19,20].

    Potential target prediction of active compounds.The potential targets for active compounds in HZKY were screened using TCMSP, TCMID, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-TCM), and PUBCHEM (https://pubchem.ncbi.nlm.nih.gov)databases.The repeated targets were counted only once.

    Potential targets for prediction of the disease.Targets related to NAFLD were obtained from GeneCards (https://www.genecards.org/) and online Mendelian Inheritance in Man (OMIM)(https://www.omim.org/) databases.The protein data bank IDs for each target were imported into the Uniprot database (http://www.uniprot.org/) for standardization,and the repeated targets were removed.In addition, the potential targets of HZKY in NAFLD were identified based on the common potential targets between HZKY and NAFLD.

    Biological function and signaling pathway analysis.The Kyoto Encyclopedia of Genes and Genomes(KEGG) database was used to integrate the information and functions of potential targets.Briefly,the potential targets were analyzed and enriched in the KEGG database (https://www.kegg.jp/).KEGG terms withP<0.05 and q <0.05 were screened.

    Establishment and analysis of the compound-target-pathway network.The relationships among compounds,targets,and pathways of HZKY in NAFLD were visualized using the compound-target-pathway network using Cytoscape 3.7.2 [21].According to the compound-target-pathway network, compounds, targets, and pathways were visualized using different colors and were combined based on their potential interactions.

    Experimental Validation

    Reagents.HFD (17.7% sucrose, 17.7% fructose,19.4% protein, and 40% fat) was purchased from Beijing HFK Bioscience Co., Ltd.(Beijing, China).A BCA protein assay kit and triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C) test kits were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing,China).Oil red O staining kit was obtained from Solarbio Biotechnology Co., Ltd.(Beijing, China).Primary antibodies against rat caspase-3 (CASP3;ab13847; 1:500), caspase-9 (CASP9; ab184786;1:1000), BAX (Bcl-2 associated X protein; ab32503;1:10000), and β-ACTIN (ab179467 1:5000) were purchased from Abcam, Inc.(Shanghai, China), and the corresponding secondary antibodies were obtained from Abcam,Inc.(Shanghai,China).

    Animals.Male Sprague-Dawley (SD) rats weighing 190-210 g, were purchased from Weitonglihua Animal Co.,Ltd.(Beijing,China).All animals were housed for 1 week in a temperature- and humidity-controlled environment (12-h light/dark cycle, 21 ± 2℃, and a relative humidity of 45 ± 10%) in groups of five rats/cage and were allowed feed and water ad libitum.

    Induction of the NAFLD model rat.The NAFLD model was induced as described previously [22].Briefly, the rats were fed with HFD diet for 12 weeks.Their body weights and serum TG, TC, LDL-C, and HDL-C levels were measured to evaluate the induction of NAFLD.In addition, H&E and oil red O staining were used to observe the pathological changes in the rat livers.All animal experiments were approved by the Institutional Animal Care and Use Committee of Tianjin University of Traditional Chinese Medicine(SYXK2019-0006) and were performed in accordance with the National Institutes of Health “Guide for the Care and Use of LaboratoryAnimals”.

    Preparation of the HZKY Test Solution.The HZKY decoction was provided by the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine(Tianjin, China).The herbs included in the HZKY decoction were identified by the pharmacist in the pharmacy of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine.The water extract of HZKY was concentrated at the laboratory of Tianjin University of Traditional Chinese Medicine.Briefly, the herbs were immersed in water for 30 min(the surface of the water covered the herbs by 2-3 cm)and boiled at 100°C for 20 min using a gallipot.The liquid was then filtered through gauze.The same volume of water and herbs were boiled for another 15 min and filtered through gauze.The two filtrates were mixed to obtain the water extract of HZKY [23].The water extract of HZKY was concentrated to a density of 0.85 g crude herb/mL under decompressed distillation using a DZF-6050 vacuum drying box(Beijing SanBo Technology Co., Ltd., Beijing, China)and SENCO R rotating evaporator (Beijing Biological Innovation Technology Co.,Ltd.,Beijing,China).

    Animal grouping.After 1 week of adaptive feeding,30 rats were randomly classified into three groups:control, model, and HZKY groups.Rats in the control group were provided standard laboratory chow containing 59.4% total carbohydrate, 20% protein, and 4.8% fat.An NAFLD model rat was induced using HFD in the model and HZKY groups.Moreover, the HZKY group received an oral gavage of HZKY(8.5 g crude herb/kg rat weight) once daily for 8 weeks after 4 weeks of HFD feeding.The dosage of 8.5 g crude herb/kg in the HZKY group was equivalent to the daily dosage of HZKY in human adults, which was calculated using the following formula: equivalent dosage (g/kg) = clinical dosage of crude herbs in humans (g/kg) × 6.25.Meanwhile, the control and model group rats were given oral gavage of same volume of saline once daily for 8 weeks after 4 weeks of feeding.

    Serum biochemical marker assays.Following HZKY treatment, rats were anesthetized (chloral hydrate intraperitoneal administration,0.3 mL/100 g rat weight)after a 5-h period of fasting, and blood was harvested from the inner canthus using a capillary glass tube.Serum was obtained by centrifugation with a SW 60 Ti rotor (Beckman Coulter Inc., Palo Alto, CA) at 3000 rpm for 15 min.Serum levels of TG, TC, LDL-C, and HDL-C were measured according to the manufacturer's instructions (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China), and the absorbance was detected using a Varioskan Flash microplate reader(Thermo Fisher Scientific,USA).

    In a 96-well plate, 2.5 μL of distilled water,TG and TC standards,and serum samples were mixed with 250 μL of working solution and incubated at 37°C for 10 min.The absorbance value of each pore was measured using a microplate reader at 510 nm.The derived optical density (OD) value of distilled water, TG and TC standards, and serum samples were denoted by OD1, OD2, and OD3, respectively.The TG and TC levels were calculated using the following formulae:

    In accordance with the instructions for LDL-C and HDL-C, 2.5 μL of distilled water, LDL-C and HDL-C standards,and serum samples were incubated with 180 μL of reagent R1 supplied by kit at 37°C for 5 min in 96-well plates.After determining the absorbance value of a microplate reader at 546 nm, the OD values of distilled water, standards, and serum samples were recorded as OD1, OD2and OD3, respectively.Then, 60 μL of reagent R2 was added into each well, repeating the previous step, and the mixed solution was incubated at 37℃for 5 min.The absorbance value was detected by microplate reader at 546 nm.The OD values of distilled water,standards,and serum samples were recorded as OD1', OD2', and OD3', respectively.The LDL-C and HDL-C levels were obtained using the following formulae:

    H&E staining.Rats were sacrificed after blood collection.The liver was immediately removed and fixed with 10%formalin,dehydrated,and embedded in paraffin wax.It was then cut into 5-μm sections using a slicing machine (RM2125, Leica, Buffalo Grove,USA) at the laboratory at Tianjin University of Traditional Chinese Medicine.Sections were stained with H&E as described previously [24].Briefly, after dewaxing, rehydration, staining, dehydration,transparency, and sealing, the pathological changes in the liver were visualized under a light microscope(BX50,Olympus America,Melville,NY,USA).

    Oil red O staining.Fresh liver tissue was rapidly frozen in liquid nitrogen and sectioned using a frozen slicer (CM3050S, Leica, Buffalo Grove, USA) to obtain coronal cryostat sections (10-μm thick).The sections were then dried at room temperature for 5 min.Next, the sections were stained with oil red O according to the manufacturer's instructions (Solarbio Biotechnology Co., Ltd., Beijing, China).Briefly, the sections were stained with oil red O lipid staining working buffer for 45 min after fixing with 5%formaldehyde solution, washed with 60% isopropanol five times, and subsequently washed with distilled water for 2 min.Sections were stained again with hematoxylin for 2 min, washed with distilled water for 5 min, and dried and sealed with glycerine.They were then immediately photographed under a light microscope (BX50, Olympus America, Melville, NY,USA).

    Western blotting analysis.The liver samples were homogenized with whole lysis buffer to extract proteins from the liver.Then,a BCA protein assay was used to quantify and standardize the proteins.Electrophoresis (8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to isolate the proteins, which were transferred onto a polyvinylidene difluoride membrane for Western blotting.The membrane was then sealed with 5% milk and incubated overnight with primary antibodies(rabbit anti-CASP1,1:500;rabbit anti-CASP9, 1:1000;rabbit anti-BAX, 1:10000; and rabbit anti-β-ACTIN,1:10000) at 4℃.After incubation for 1 hour, TBST was used to clean the membrane, and the secondary antibody probe (HRP combined with goat anti-rabbit IgG, 1:4000) was used for detection.Blotting was observed using chemiluminescence (TanonTMHigh-sig ECL, Li COR, USA).The strip strength was quantitatively analyzed using Image J software (NIH,Bethesda,MD,USA).

    Data Analysis.All values were reported as the mean±standard deviation (mean ± SD).SPSS software version 20.0 was used for single factor analysis of variance, and the statistical significance standard wasP<0.05.

    Results

    Results of Network Pharmacology

    Main active compounds in HZKY.The 924 compounds present in HZKY, including 65 from Zhishi (Aurantii Fructus Immaturus), 60 from Peilan(Eupatorii Herba), 58 from Huangqin (Scutellariae Radix), 93 from Heye (Nelumbinis Folium), 92 from Dahuang (Rhei Radix et Rhizoma), 75 from Shaoyao(Paeoniae Radix Rubra), 288 from Chaihu (Bupleuri Radix), 77 from Bohe (Menthae Haplocalycis Herba),and 116 from Banxia (Pinelliae Rhizoma), were screened using the TCMSP and TCMID.A total of 130 active compounds, including 22 from Zhishi (Aurantii Fructus Immaturus),11 from Peilan(Eupatorii Herba),24 from Huangqin (Scutellariae Radix), 15 from Heye(Nelumbinis Folium), 16 from Dahuang (Rhei Radix et Rhizoma), 27 from Shaoyao (Paeoniae Radix Rubra),17 from Chaihu (Bupleuri Radix), 10 from Bohe(Menthae Haplocalycis Herba), and 36 from Banxia(Pinelliae Rhizoma), were identified with OB ≥ 30%and DL ≥ 0.18.The structure and physiochemical properties of the compounds are listed in Table 1.

    Table 1 List of active candidate components in HZKY

    Table 1 List of active candidate components in HZKY(Continued)

    Table 1 List of active candidate components in HZKY(Continued)

    Potential targets of active compounds in HZKY.A total of 1,672 targets were obtained from 130 active compounds in HZKY based on the TCMSP, TCMID,Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and PUBCHEM databases.Repeated targets were removed,and 269 targets were identified with alignment from the Uniprot database.

    Identification of potential targets and pathways of HZKY in NAFLD and network analysis of compounds, targets, and pathways.A total of 1,334 targets for NAFLD were identified from the online Mendelian Inheritance in Man and GeneCards databases.After intersecting with 269 targets of HZKY,62 targeted were identified as potential targets of HZKY in NAFLD.

    The interactive relationship of 62 potential targets was analyzed and visualized using String 11.0.The candidates with the highest values of network connectivity were BAX, CASP3, CASP9, IL6, EGFR,MAPK8, CYCS, ESR1, VEGFA, LDLR, PPARG,PLAU, VCAM1, CYP3A4, GSK3B, PON1, GSTP1,NQO1, CTSD, and ACACA.BAX, CASP3, and CASP9 were the targets with the highest values of network connectivity.Additionally, as determined using KEGG analysis, apoptosis, fatty acid synthesis,and estrogen signaling pathways were the terms with the highest network connectivity.

    The compounds, targets, and pathways of HZKY in NAFLD were imported into Cytoscape 3.7.2 to establish the compound-target-pathway network(Figure 1).

    According to the compound-target-pathway network,compounds targeted to BAX included M56, M95, and M116; compounds targeted to CASP9 included M1,M2, M3, M5, M8, M9, M10, M13, M14, M21, M25,M29, M31, M33, M34, M35, M39, M49, M50, M57,M59, M62, and M125; and compounds targeted to CASP3 included M1, M2, M3, M4, M5, M6, M8, M9,M10, M14, M13, M19, M21, M23, M25, M33, M34,M35, M38, M39, M49, M50, M56, M57, M58, M59,M62,M64,M65,M84,M95,M116,M125,and M130.Moreover, BAX, CASP9, and CASP3 are closely associated with the apoptosis pathway.Therefore, the apoptosis pathway was predicted to be the potential mechanism of action of HZKY in NAFLD.

    Results of Experimental Validation

    HZKY ameliorated HFD-induced NAFLD in rats.Using the NAFLD model rat, we observed that 12 weeks of HFD led to a significant increase in body weight in the model group compared with that in the control group (467.07 ± 15.31 vs.414.29 ± 28.93 g,P< 0.01), whereas HZKY treatment significantly decreased the body weight in the HZKY group compared with that in the model group (420.57 ±15.52 vs.467.07 ± 15.31 g,P<0.01, Table 2).In addition,the serum levels of TG,TC,and LDL-C were significantly elevated and the serum level of HDL-C was decreased in the model group compared with that in the control group (TG: 1.70 ± 0.13 vs.0.28 ± 0.04,TC: 2.58 ± 0.34 vs.1.40 ± 0.26, LDL-C: 1.50 ± 0.23 vs.0.89 ± 0.09, and HDL-C: 1.29 ± 0.16 vs.1.86 ±0.18,P<0.01 for all, Table 2).HZKY treatment decreased the serum levels of TG,TC, and LDL-C and increased the serum level of HDL in the HZKY group compared with those in the model group (TC: 2.16 ±0.22 vs.2.58±0.34,P<0.01;TG:1.28±0.16 vs.1.70± 0.13,P<0.05; LDL-C: 1.14 ± 0.22 vs.1.50 ± 0.23,P<0.05;and HDL-C:1.49±0.14 vs.1.29±0.16,P<0.05,Table 2).

    H&E staining demonstrated that the hepatic lobule and nucleus were clearly visible, the sizes of the hepatocytes were similar, and the hepatic cord was arranged neatly in the control group.Compared with that in the control group, in the model group, hepatic steatosis was obvious, indicating that there were vacuolar adipose droplets of various sizes and more balloon-like changes in hepatocytes, and the structure of the hepatic tissue could not be clearly observed.HZKY treatment markedly alleviated the hepatic steatosis and balloon-like changes in the HZKY group compared with that in the model group(Figure 2a).

    Similarly, oil red O staining revealed that slight lipid deposition was observed in the control group.The lipid droplets in the cytoplasm of hepatocytes were stained red and were of various sizes and widely distributed in the cytoplasm, indicating the presence of lipid deposition in a large number of hepatocytes.However,HZKY significantly ameliorated lipid deposition and reduced the number of intracellular lipid droplets(Figure 2b).

    Figure 1 Compound-target-pathway network.The blue nodes represent the targets, the yellow nodes represent the active components of HZKY,and the red nodes represent pathways.Every active ingredient can act on multiple targets.

    Table 2 HZKY treatment decreased body weight and changed the blood lipid composition in NAFLD model rats.

    HZKY inhibited apoptosis in NAFLD model rats

    According to pathways identified from the network pharmacology, apoptosis was selected as the mechanism for HZKY in NAFLD.Expressions of BAX, CASP9, and CASP3, which were predicted as the top targets of HZKY in NAFLD, were investigated to determine the anti-apoptotic activities of HZKY in NAFLD.The expressions of BAX, CASP9, and CASP3 were elevated in the model group compared with those in the control group(for BAX,P<0.05;for CASP9 and CASP3,P<0.01; Figure 3), whereas HZKY treatment decreased the expressions of BAX,CASP9, and CASP3 in the HZKY group compared with those the model group(for BAX and CASP9,P<0.05;for CASP3,P<0.01;Figure 3).

    Discussions

    In this study,the potential targets of HZKY in NAFLD were predicted using network pharmacology.Many compounds in HZKY, such as baicalein,o-nornuciferine, and quercetin, have been demonstrated to have treatment effects in NAFLD.Baicalein, derived from Banxia (Pinelliae Rhizoma),Huangqin (Scutellariae Radix), and Shaoyao(Paeoniae Radix Alba), is shown to reduce lipid accumulation in NAFLD model rats by inhibiting oxidative stress [25].O-Nornuciferine, derived from Heye (Nelumbinis Folium), decreases the serum TG and TC levels in NAFLD mouse models [26].Quercetin, derived from Chaihu (Bupleuri Radix) and Heye (Nelumbinis Folium), improves NAFLD via downregulation of FAS and SREBP-1C expression and upregulation of IRS1 expression in the liver [27].In addition, BAX, CASP9, and CASP3 were the HZKY targets with the highest values of network connectivity,and apoptosis was the main signaling pathway of HZKY in NAFLD.The effects of HZKY in NAFLD model rats were also investigated in the present study.The results showed that HZKY treatment significantly improved the serum levels of TG, TC, LDL-C, and HDL-C in NAFLD model rats.Moreover, lipid accumulation in the liver was reduced following HZKY treatment, indicating the significant curative effect of HZKY in NAFLD.

    NAFLD is accompanied by the apoptosis of hepatocytes [28]; electron microscopy previously showed that the ultrastructures of mitochondria were damaged in the hepatocytes of patients with NAFLD[29].Steatosis in hepatocytes can impair the function and decrease the membrane potential of mitochondria.A decrease in the mitochondrial membrane potential can trigger the release of cytochrome C and activate caspase family proteins, ultimately inducing apoptosis of hepatocytes [30].Furthermore, dysfunction in fatty acid synthesis can cause obesity and NAFLD.The uptake and synthesis of fatty acids in the liver is significantly increased in the liver [31].Excessive amounts of fatty acid can trigger IR by inducing oxidative stress and decreasing sensitivity in hepatocytes.IR can cause dysfunction of lipase activity,thereby triggering lipolysis in the peripheral organs and increasing the levels of fatty acids in the serum[32].Moreover,IR induces hyperinsulinemia, and high levels of insulin inhibit the β-oxidation of fatty acids.The excess fatty acids can be transported to the liver and cause steatosis in hepatocytes and NAFLD [33].Estrogen has also been demonstrated to be associated with NAFLD.Compared with premenopausal women,postmenopausal women exhibit an increase in body weight, lipid accumulation, hepatic inflammatory responses, and oxidative stress [34].The activation of estrogen receptors can increase the production of bile acid in NAFLD model mice.Bile acid can activate the farnesoid X receptor to inhibit lipogenesis in the liver.Estrogen receptors can also directly reduce the lipid contents and liver injury in NAFLD model mice by inhibiting the activation of pregnane X receptor.Estrogen can also improve NAFLD by regulating the TLR-MYD88-IL-6 pathway.Estrogen has been demonstrated to improve hepatocyte injury in NAFLD by inhibiting the activation of NF-κB and C/EBPβ and decreasing the activity of IL-6 promoter [35] .In addition, lack of estrogen can contribute to the progression of NAFLD[36-37].BAX,CASP9, and CASP3 are important factors that modulate apoptosis in NAFLD.Accumulation of free fatty acids is shown to activate and dimerize BAX in the cytoplasm [38].Dimerized BAX is transported to the outer membrane of the mitochondria and activates CASP-9 and CASP-3 to initiate apoptosis[39].

    Given the high correlation of the apoptotic pathway predicted by network pharmacology, we determined the anti-apoptotic activities of HZKY in NAFLD model rats.In agreement with the results of network pharmacology analysis, our results show that HZKY treatment significantly decrease the levels of apoptosis-related proteins, including BAX, CASP9,and CASP3,in the liver.

    Figure 2 Hepatic steatosis was improved after HZKY treatment.

    Figure 3 HZKY treatment regulated the expressions of BAX,CASP9 and CASP3 in NAFLD model rats.

    Conclusion

    The present study demonstrates that HZKY improves NAFLD by inhibiting apoptosis in the liver by decreasing the levels of apoptosis-related proteins,including BAX,CASP9,and CASP3.

    免费观看无遮挡的男女| 国产欧美亚洲国产| 一区二区三区精品91| 中文精品一卡2卡3卡4更新| 好男人视频免费观看在线| 欧美丝袜亚洲另类| 日韩在线高清观看一区二区三区| av在线观看视频网站免费| 国产亚洲欧美精品永久| 国内精品宾馆在线| 国产日韩欧美在线精品| 三级国产精品欧美在线观看| 国产精品国产三级国产av玫瑰| 日日摸夜夜添夜夜爱| 婷婷色av中文字幕| 欧美激情极品国产一区二区三区 | 国产极品粉嫩免费观看在线 | 成年女人在线观看亚洲视频| 一级毛片黄色毛片免费观看视频| 在线观看三级黄色| 黄色视频在线播放观看不卡| 国产精品国产av在线观看| 满18在线观看网站| 一级a做视频免费观看| 我要看黄色一级片免费的| 精品亚洲乱码少妇综合久久| 在线天堂最新版资源| www.色视频.com| 精品少妇内射三级| 伊人久久国产一区二区| 国产精品久久久久久久电影| 国产欧美另类精品又又久久亚洲欧美| 亚洲精品日本国产第一区| 少妇精品久久久久久久| 纵有疾风起免费观看全集完整版| 免费av中文字幕在线| 久久久久精品性色| 少妇被粗大的猛进出69影院 | 亚洲av中文av极速乱| 国产精品免费大片| 亚洲欧美一区二区三区国产| 热99久久久久精品小说推荐| 欧美日韩视频精品一区| 卡戴珊不雅视频在线播放| 久久久精品区二区三区| 少妇 在线观看| 交换朋友夫妻互换小说| 日韩一本色道免费dvd| 国产精品 国内视频| 如日韩欧美国产精品一区二区三区 | 久久久久久久亚洲中文字幕| 赤兔流量卡办理| 久久鲁丝午夜福利片| 成人综合一区亚洲| 国产色婷婷99| 国产在线视频一区二区| 久久国产精品大桥未久av| 国产精品.久久久| 肉色欧美久久久久久久蜜桃| 中文字幕人妻丝袜制服| 国产有黄有色有爽视频| 中文字幕人妻丝袜制服| 久久99一区二区三区| 久久免费观看电影| 国产午夜精品久久久久久一区二区三区| 精品国产乱码久久久久久小说| 天美传媒精品一区二区| 亚洲人成网站在线播| 性高湖久久久久久久久免费观看| 亚洲av.av天堂| 国产成人a∨麻豆精品| 久久精品国产a三级三级三级| 夫妻性生交免费视频一级片| 国产亚洲欧美精品永久| 久久99热6这里只有精品| 国产精品人妻久久久久久| 这个男人来自地球电影免费观看 | 五月开心婷婷网| 日本wwww免费看| 久久午夜福利片| 极品少妇高潮喷水抽搐| 久久人妻熟女aⅴ| 热re99久久精品国产66热6| 亚洲第一av免费看| 久久久亚洲精品成人影院| 91久久精品国产一区二区三区| 亚洲人与动物交配视频| 午夜视频国产福利| 国产精品成人在线| 啦啦啦中文免费视频观看日本| 香蕉精品网在线| 国产成人精品福利久久| 亚洲精品色激情综合| 免费日韩欧美在线观看| 一区二区日韩欧美中文字幕 | 亚洲怡红院男人天堂| 99九九在线精品视频| 精品亚洲成a人片在线观看| 熟女电影av网| 欧美另类一区| 午夜精品国产一区二区电影| 国产精品久久久久久久电影| 91久久精品国产一区二区三区| 久久久精品区二区三区| 女性生殖器流出的白浆| 在线观看三级黄色| 国产精品一二三区在线看| 亚洲久久久国产精品| 亚洲精品,欧美精品| 黑人高潮一二区| 51国产日韩欧美| 亚洲欧美中文字幕日韩二区| 亚洲第一av免费看| 久久久久久久久久久免费av| 少妇被粗大猛烈的视频| av在线播放精品| 99热这里只有是精品在线观看| 久久狼人影院| 人人妻人人爽人人添夜夜欢视频| 大话2 男鬼变身卡| 国产精品人妻久久久影院| 一边亲一边摸免费视频| 国产成人免费无遮挡视频| 性色avwww在线观看| 啦啦啦啦在线视频资源| 亚洲三级黄色毛片| 精品国产一区二区三区久久久樱花| av播播在线观看一区| 久久久久久久久大av| av在线播放精品| 蜜桃国产av成人99| 亚洲无线观看免费| 亚洲精品视频女| 久久精品国产自在天天线| 999精品在线视频| 999精品在线视频| 日韩,欧美,国产一区二区三区| 纵有疾风起免费观看全集完整版| 色吧在线观看| 视频中文字幕在线观看| 国产精品熟女久久久久浪| av黄色大香蕉| 你懂的网址亚洲精品在线观看| 国产亚洲欧美精品永久| 狂野欧美激情性xxxx在线观看| 人妻系列 视频| 一个人看视频在线观看www免费| 久久精品国产自在天天线| 国产精品久久久久久av不卡| 国产一级毛片在线| 人妻一区二区av| 国产成人精品久久久久久| 一级毛片 在线播放| 18禁在线无遮挡免费观看视频| 亚洲精品中文字幕在线视频| 午夜免费男女啪啪视频观看| 2022亚洲国产成人精品| 日韩大片免费观看网站| 99久国产av精品国产电影| 国产日韩欧美在线精品| 午夜福利在线观看免费完整高清在| 女人精品久久久久毛片| 亚洲高清免费不卡视频| 如何舔出高潮| 日韩,欧美,国产一区二区三区| 国产 一区精品| 在线观看三级黄色| 午夜免费鲁丝| 日日摸夜夜添夜夜爱| 亚洲av电影在线观看一区二区三区| 亚洲国产精品一区三区| 黄色怎么调成土黄色| 在线观看美女被高潮喷水网站| 免费观看性生交大片5| 卡戴珊不雅视频在线播放| 黄色毛片三级朝国网站| 在线 av 中文字幕| 成人二区视频| 国产成人精品一,二区| 国产成人一区二区在线| 最新的欧美精品一区二区| 有码 亚洲区| 麻豆乱淫一区二区| 日韩成人伦理影院| 亚洲激情五月婷婷啪啪| 国产精品女同一区二区软件| 99热这里只有精品一区| 国产av码专区亚洲av| 日本wwww免费看| av国产精品久久久久影院| 美女cb高潮喷水在线观看| 欧美成人精品欧美一级黄| 欧美变态另类bdsm刘玥| a级毛片在线看网站| 五月玫瑰六月丁香| 最近2019中文字幕mv第一页| 99久久精品一区二区三区| 成人毛片60女人毛片免费| 男女边吃奶边做爰视频| 日本欧美国产在线视频| 精品亚洲乱码少妇综合久久| 国产白丝娇喘喷水9色精品| av福利片在线| 人人澡人人妻人| 免费日韩欧美在线观看| 丝袜脚勾引网站| 免费看av在线观看网站| 一个人看视频在线观看www免费| 久久热精品热| 亚洲美女黄色视频免费看| 欧美日韩视频高清一区二区三区二| 丝袜脚勾引网站| 亚洲欧美色中文字幕在线| 在线精品无人区一区二区三| 欧美精品一区二区免费开放| 国产免费视频播放在线视频| 国产视频内射| 免费看光身美女| 国产午夜精品久久久久久一区二区三区| 免费观看无遮挡的男女| 亚洲少妇的诱惑av| 国产不卡av网站在线观看| av在线观看视频网站免费| 人妻人人澡人人爽人人| 黄色配什么色好看| 亚洲国产精品一区三区| 99久久精品一区二区三区| 搡女人真爽免费视频火全软件| 狂野欧美白嫩少妇大欣赏| videosex国产| 久久精品久久精品一区二区三区| 日本91视频免费播放| 欧美精品一区二区免费开放| 免费黄色在线免费观看| 三级国产精品欧美在线观看| 一区二区三区乱码不卡18| a级片在线免费高清观看视频| 日日撸夜夜添| 九九爱精品视频在线观看| 视频在线观看一区二区三区| 99九九在线精品视频| 久久av网站| 精品午夜福利在线看| 中文字幕亚洲精品专区| 亚洲精品视频女| 日韩免费高清中文字幕av| 国产在线视频一区二区| 夫妻午夜视频| 国产乱人偷精品视频| 亚洲精品久久久久久婷婷小说| videossex国产| 伦精品一区二区三区| 精品久久蜜臀av无| 久久精品国产亚洲av天美| 一区二区三区精品91| 在线 av 中文字幕| 人人妻人人澡人人爽人人夜夜| 精品一区在线观看国产| 夜夜爽夜夜爽视频| 亚洲一级一片aⅴ在线观看| 晚上一个人看的免费电影| 国产精品 国内视频| videossex国产| 国产探花极品一区二区| 国产精品三级大全| 国产免费又黄又爽又色| 一级毛片 在线播放| 晚上一个人看的免费电影| 一级,二级,三级黄色视频| 日韩欧美精品免费久久| 国产成人freesex在线| 夫妻午夜视频| 免费少妇av软件| 一区二区av电影网| 国内精品宾馆在线| 99热这里只有精品一区| 一区二区三区免费毛片| 欧美3d第一页| 欧美三级亚洲精品| 亚洲精品久久午夜乱码| 美女中出高潮动态图| 日韩伦理黄色片| 97精品久久久久久久久久精品| 夫妻性生交免费视频一级片| 久久影院123| 国产高清三级在线| 日本av免费视频播放| 老司机亚洲免费影院| 国产一区二区三区综合在线观看 | 精品久久国产蜜桃| 纯流量卡能插随身wifi吗| 亚洲激情五月婷婷啪啪| 最后的刺客免费高清国语| 久久久久久久久久成人| 国产国拍精品亚洲av在线观看| 内地一区二区视频在线| 国产精品国产三级专区第一集| 精品久久久精品久久久| 91成人精品电影| 精品熟女少妇av免费看| 极品人妻少妇av视频| 大片免费播放器 马上看| 丁香六月天网| 成人毛片60女人毛片免费| √禁漫天堂资源中文www| 亚洲精品成人av观看孕妇| 国产探花极品一区二区| 日本wwww免费看| 一本久久精品| 欧美一级a爱片免费观看看| 永久免费av网站大全| 国产综合精华液| 精品99又大又爽又粗少妇毛片| 亚洲成人av在线免费| 国产国拍精品亚洲av在线观看| a级毛色黄片| 伦理电影大哥的女人| 亚州av有码| 高清在线视频一区二区三区| 国产成人91sexporn| 熟妇人妻不卡中文字幕| 亚洲欧洲国产日韩| 亚洲av在线观看美女高潮| 观看av在线不卡| 丰满饥渴人妻一区二区三| 午夜日本视频在线| 午夜激情福利司机影院| 这个男人来自地球电影免费观看 | 美女主播在线视频| 欧美亚洲日本最大视频资源| 婷婷色综合大香蕉| 精品99又大又爽又粗少妇毛片| 日本色播在线视频| 国产精品蜜桃在线观看| 3wmmmm亚洲av在线观看| 免费黄网站久久成人精品| 日韩中字成人| 蜜桃久久精品国产亚洲av| 亚洲熟女精品中文字幕| 欧美 日韩 精品 国产| 欧美精品一区二区免费开放| 51国产日韩欧美| 黄色配什么色好看| 亚洲精品日韩av片在线观看| 欧美精品一区二区大全| 亚洲美女视频黄频| 久久精品夜色国产| 亚洲性久久影院| 美女主播在线视频| 免费观看的影片在线观看| 久久国产精品大桥未久av| 国产精品久久久久久av不卡| 大陆偷拍与自拍| 精品卡一卡二卡四卡免费| 成人亚洲精品一区在线观看| 丝袜美足系列| 国产男女超爽视频在线观看| av电影中文网址| 在线天堂最新版资源| 性色avwww在线观看| 99精国产麻豆久久婷婷| 高清欧美精品videossex| 成人毛片60女人毛片免费| 亚洲欧美日韩卡通动漫| 女人久久www免费人成看片| 久久97久久精品| 岛国毛片在线播放| 一区二区三区免费毛片| 亚洲第一区二区三区不卡| 大香蕉97超碰在线| 纵有疾风起免费观看全集完整版| 免费黄色在线免费观看| 国产午夜精品久久久久久一区二区三区| 天堂俺去俺来也www色官网| 91国产中文字幕| 国产精品久久久久久精品古装| 国产 一区精品| 国产精品久久久久久久久免| av福利片在线| 极品少妇高潮喷水抽搐| 中文精品一卡2卡3卡4更新| 亚洲三级黄色毛片| 男女高潮啪啪啪动态图| 伦精品一区二区三区| 校园人妻丝袜中文字幕| 成人免费观看视频高清| 婷婷色综合www| 你懂的网址亚洲精品在线观看| 一本大道久久a久久精品| 中文字幕人妻丝袜制服| 国产精品一区www在线观看| 美女大奶头黄色视频| 国产精品99久久99久久久不卡 | 国产老妇伦熟女老妇高清| 高清黄色对白视频在线免费看| 热99国产精品久久久久久7| 男女免费视频国产| 亚洲欧美日韩另类电影网站| 欧美精品一区二区免费开放| 熟妇人妻不卡中文字幕| 亚洲精品乱久久久久久| 久久久精品免费免费高清| 欧美另类一区| 高清视频免费观看一区二区| 校园人妻丝袜中文字幕| 久久久国产精品麻豆| 一区二区三区免费毛片| 99视频精品全部免费 在线| 亚洲欧美清纯卡通| 大话2 男鬼变身卡| 51国产日韩欧美| 日产精品乱码卡一卡2卡三| 天堂俺去俺来也www色官网| 国产精品一国产av| 欧美成人午夜免费资源| 久久久久久久久大av| 少妇精品久久久久久久| 国产亚洲午夜精品一区二区久久| 国产精品人妻久久久久久| 人人澡人人妻人| 九草在线视频观看| 高清欧美精品videossex| 天天操日日干夜夜撸| 少妇熟女欧美另类| 婷婷色综合大香蕉| 老女人水多毛片| 又粗又硬又长又爽又黄的视频| 一区二区日韩欧美中文字幕 | 成人无遮挡网站| 午夜福利在线观看免费完整高清在| 下体分泌物呈黄色| 欧美精品一区二区免费开放| 亚洲精品乱码久久久v下载方式| 色吧在线观看| av视频免费观看在线观看| 丰满迷人的少妇在线观看| 国产欧美亚洲国产| 中文字幕精品免费在线观看视频 | 欧美另类一区| 国产一区二区三区综合在线观看 | 乱码一卡2卡4卡精品| 欧美精品国产亚洲| 亚洲国产精品999| 91在线精品国自产拍蜜月| 国产高清不卡午夜福利| a 毛片基地| 久久99精品国语久久久| 中文字幕亚洲精品专区| 国产欧美另类精品又又久久亚洲欧美| 国产不卡av网站在线观看| av国产精品久久久久影院| 亚洲成人av在线免费| 成年人午夜在线观看视频| 蜜臀久久99精品久久宅男| 亚洲精品中文字幕在线视频| 国产成人免费观看mmmm| 精品一品国产午夜福利视频| 亚洲情色 制服丝袜| 久久精品人人爽人人爽视色| 中文字幕精品免费在线观看视频 | 青春草亚洲视频在线观看| 多毛熟女@视频| 国产不卡av网站在线观看| 日本av手机在线免费观看| 波野结衣二区三区在线| 在线 av 中文字幕| 国产精品无大码| 久久精品国产鲁丝片午夜精品| 亚洲av福利一区| 亚洲精华国产精华液的使用体验| 免费观看无遮挡的男女| 大话2 男鬼变身卡| 国产无遮挡羞羞视频在线观看| 视频区图区小说| 国产日韩欧美在线精品| 免费大片18禁| 国产有黄有色有爽视频| 一边摸一边做爽爽视频免费| 日本wwww免费看| 国产淫语在线视频| 国产成人av激情在线播放 | 久久99热这里只频精品6学生| 免费av不卡在线播放| 老司机影院成人| 一区二区三区精品91| 久久精品国产a三级三级三级| 少妇的逼好多水| 欧美人与善性xxx| 精品久久久精品久久久| 2022亚洲国产成人精品| 久久精品国产鲁丝片午夜精品| 免费久久久久久久精品成人欧美视频 | 精品亚洲成国产av| 成人影院久久| 成人亚洲精品一区在线观看| 熟女人妻精品中文字幕| 最新中文字幕久久久久| 成人综合一区亚洲| 精品少妇久久久久久888优播| 黑丝袜美女国产一区| 欧美精品亚洲一区二区| 国产国语露脸激情在线看| 男女无遮挡免费网站观看| 天美传媒精品一区二区| 亚洲欧美精品自产自拍| 999精品在线视频| 亚洲精品乱久久久久久| 人人妻人人爽人人添夜夜欢视频| 一本久久精品| 日本与韩国留学比较| 中文字幕人妻熟人妻熟丝袜美| 丰满乱子伦码专区| 五月开心婷婷网| 国产精品久久久久久精品电影小说| 一区在线观看完整版| 免费黄频网站在线观看国产| 99九九线精品视频在线观看视频| 插逼视频在线观看| 麻豆乱淫一区二区| 一级毛片 在线播放| 亚洲精品一二三| av卡一久久| 人成视频在线观看免费观看| 18禁动态无遮挡网站| 国产成人精品婷婷| 日韩,欧美,国产一区二区三区| 免费日韩欧美在线观看| 婷婷色综合大香蕉| 久久精品熟女亚洲av麻豆精品| av在线播放精品| 永久免费av网站大全| 亚洲国产成人一精品久久久| 少妇的逼好多水| 国产成人精品福利久久| 亚洲欧美一区二区三区国产| 国产极品粉嫩免费观看在线 | 亚洲av男天堂| 亚洲精品一二三| 大片免费播放器 马上看| 在线天堂最新版资源| 五月天丁香电影| 久久久亚洲精品成人影院| 在线免费观看不下载黄p国产| 精品人妻偷拍中文字幕| 三级国产精品片| 91aial.com中文字幕在线观看| 一级黄片播放器| 午夜av观看不卡| 国产精品久久久久久久久免| av福利片在线| 久久精品国产亚洲av天美| av免费在线看不卡| 久久国产精品男人的天堂亚洲 | 亚洲av电影在线观看一区二区三区| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 国产亚洲精品第一综合不卡 | 亚洲欧洲精品一区二区精品久久久 | 亚洲欧美精品自产自拍| 国产精品久久久久久久久免| 日韩av免费高清视频| 久久久久久久久久久免费av| 一边摸一边做爽爽视频免费| 中国三级夫妇交换| 国产一区二区三区综合在线观看 | 亚洲人成网站在线观看播放| 能在线免费看毛片的网站| av播播在线观看一区| 黄色欧美视频在线观看| 少妇猛男粗大的猛烈进出视频| 亚洲精品乱码久久久久久按摩| av黄色大香蕉| 成人漫画全彩无遮挡| 大香蕉久久网| 人妻人人澡人人爽人人| 2022亚洲国产成人精品| 国产高清三级在线| 在线观看美女被高潮喷水网站| videosex国产| 国产老妇伦熟女老妇高清| 国产精品一国产av| 一区二区日韩欧美中文字幕 | 免费黄色在线免费观看| 亚洲一级一片aⅴ在线观看| 十八禁网站网址无遮挡| 欧美日韩av久久| 99热网站在线观看| 国产探花极品一区二区| 午夜激情av网站| 亚洲av.av天堂| 不卡视频在线观看欧美| 最近2019中文字幕mv第一页| 色吧在线观看| 九草在线视频观看| 99热全是精品| 日韩三级伦理在线观看| 国产精品.久久久| 国产精品一二三区在线看| 亚洲综合色网址| 人人妻人人澡人人爽人人夜夜| 久久99热6这里只有精品| 一级片'在线观看视频| 亚洲av二区三区四区| 欧美丝袜亚洲另类| 午夜久久久在线观看| 熟女电影av网| 国产欧美日韩一区二区三区在线 | 国产精品一区二区在线观看99| 在线观看www视频免费| 色哟哟·www| 日本免费在线观看一区| 成年av动漫网址| 久久综合国产亚洲精品| 伊人亚洲综合成人网| 久久久a久久爽久久v久久| 久久国产亚洲av麻豆专区|