• <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 ?

    Comparative study of the hemolytic and cytotoxic activities of nematocyst venoms from the jelly fish Cyanea nozakii Kishinouye and Nemopilema nomurai Kishinouye*

    2018-08-02 02:50:58PANGMin龐敏XUJintao徐金濤LIUYunlong劉云龍ZHANGXuelei張學(xué)雷
    Journal of Oceanology and Limnology 2018年4期

    PANG Min (龐敏) , XU Jintao (徐金濤) , LIU Yunlong (劉云龍) , ZHANG Xuelei (張學(xué)雷) ,

    1 . Key Laboratary of Science and Engineering for Marine Ecology and Environment, First Institute of Oceanography, State Oceanic Administration, Qingdao 266003, China

    2 . Laboratory of Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology,Qingdao 266200,China

    3 . Qinhuangdao Marine Environmental Monitoring Central Station of SOA, Qinhuangdao 066000, China

    4 Chinese Research Academy of Environmental Sciences, Beijing 100012, China

    Abstract Two species of jelly fish, Cyanea nozakii Kishinouye and Nemopilema nomurai Kishinouye,have occurred off coastal areas of the northeastern China Sea, Yellow Sea, and Bohai Sea in recent years. They in fluence marine ecosystem safety and fishery production, and also pose a risk to human health. The current study examined the hemolytic and cytotoxic activities of crude venoms extracted from the nematocysts of C.nozakii and N. nomurai. The results showed that there were more nematocysts on tentacles from C. nozakii than on tentacles of the same length from N. nomurai. The protein concentration per nematocyst extracted from N. nomurai was higher than that from C. nozakii. Both nematocyst venoms showed dose- and timedependent hemolytic activity on erythrocytes from chicken, pigeon, and sheep, with sheep erythrocytes being the most sensitive, with EC 50 values of 69.69 and 63.62 μg/mL over a 30-min exposure with N.nomurai and C. nozakii nematocyst venoms, respectively. A cytotoxic assay of both jelly fish venoms on A431 human epidermal carcinoma cells resulted in IC 50 values of 68.6 and 40.9 μg/mL after 24-h incubation,respectively, with venom from C. nozakii showing stronger cytotoxic activity than that from N. nomurai.The results of current study indicate that nematocyst venom from C. nozakii had stronger hemolytic and cytotoxic activities than that from N. nomurai and, thus, C. nozakii might be more harmful to the health of humans and other species than are N. nomurai when they appear in coastal waters.

    Keyword: Cyanea nozakii; Nemopilema nomurai; nematocyst venom; jelly fish; hemolytic activity;cytotoxicity

    1 INTRODUCTION

    Blooms of the jelly fish Cyanea nozakii Kishinouye( C. nozakii), a cnidarian of the Class Scyphomedusae,Order Semaeostomeae, Family Cyaneidae, have occurred off coastal areas of the Northeastern China Sea, Yellow Sea, and Bohai Sea since the late 20th century (Dong et al., 2006). In addition, blooms of the jelly fish Nemopilema nomurai Kishinouye( N. nomurai), a cnidarian of the Class Scyphomedusae,Order Rhizostomeae, Family Stomolophidae (Huang and Lin, 2012) have occurred in recent years in temperate China seas, including the northern East China Sea, Yellow Sea, and Bohai Sea (Dong et al.,2010). Both C. nozakii and N. nomurai, which are often abundant in late summer to early autumn from the Bohai Sea to the Yellow Sea, are the major species causing blooms in the northern China seas. Such blooms have deleterious effects on marine ecosystems as well as fishery production, and pose a risk to human health. Humans stung by either species suffer itching,edema, muscle aches, tightness of breath, hypotension,shock, and even death (Yu et al., 2005; Li et al., 2013,2014). These symptoms occur in response to the venom contained in nematocysts on the tentacles of the jelly fish. These venoms contain toxic polypeptides and/or proteins, which all increase the risk to human health during jelly fish blooms.

    Many biological assays have been carried out to estimate the toxicity of several jelly fish venoms.Hemolytic proteins in nematocyst venoms are key markers of the toxic effects of jelly fish stings,including erythema and edema (Li et al., 2013);therefore, hemolytic and cytotoxic activities of jelly fish venoms have been the most widely reported(Nagai et al., 2000a, b; Brinkman and Burnell, 2007;Helmholz et al., 2007; Yu et al., 2007; Marino et al.,2008, 2009). The hemolytic and cytotoxic activities of venoms from C. nozakii and N. nomurai have also been evaluated. Kang et al. (2009) assessed the hemolytic activity of N. nomurai venom on cat, dog,human, rabbit, and rat erythrocytes, and evaluated its cytotoxicity using C2C12 (skeletal myoblast) and H9C2 (heart myoblast) cell lines. Feng et al. (2010)partially characterized the hemolytic activity of nematocyst venom from C. nozakii on chicken erythrocytes. Lee et al. (2011) compared the cytotoxicity of venoms extracted from N. nomurai,Rhopilema esculenta, C. nozakii, and Aurelia aurita on NIH 3T3 mouse fibroblast cells. Li et al. (2012)investigated the cytotoxicity of C. nozakii on Bel-7402 and SMMC-7721 human hepatoma cells and on H630 human colon cancer cells. However, as far as we are aware, there is no published report comparing the activity intensity of venoms from C. nozakii and N. nomurai. Therefore, in the current study, we compared the toxic activities of crude venoms from C. nozakii and N. nomurai nematocysts using hemolytic and cytotoxic assays. The results provide a novel viewpoint for understanding the threats to human health posed by these two jelly fish species.

    2 MATERIAL AND METHOD

    2.1 Preparation of nematocysts

    Specimens of C. nozakii and N. nomurai were collected from the near-shore area in Qingdao, China,in August 2012, and transferred to the laboratory immediately. All tentacles were removed from living jelly fish bodies, no less than ten tentacles were selected at random, and the remaining tentacles were stored at 80° C before use. The selected tentacles were unbended on aluminum foils separately and left stretching naturally at 4℃ for 5 min. Then three 5 mm-long sections were cut randomly from each tentacle and transferred to one drop of sterilized and filtered seawater on the glass slide under microscope to count the numbers of nematocysts. All sections cut from selected tentacles of jelly fish specimens were observed, and the distribution and density of nematocysts in clusters on the hollow tubular tentacles were estimated totally.

    Nematocysts were isolated from frozen tentacles following the methods described previously (Bloom et al., 1998) with a slight modi fication. Brie fly, frozen tentacles were thawed at 4° C and immersed for 3 days in a volume of sterilized and filtered (0.45 μm) natural seawater that was twice that of the tissue volume to enable autolysis. The mixture was stirred gently twice daily. The resulting suspension was filtered using different-sized mesh (following the order 250 μm,160 μm, 90 μm, and 40 μm), and washed three times with sterilized and filtered natural seawater. After each round of washing, the filtrate was centrifuged at 3 412× g at 4° C for 20 min and the debris was removed with a pipette. All The final undischarged nematocysts were collected, examined microscopically, and then stored at -80°C until further use.

    2.2 Venom extraction and preparation

    Venom was extracted from nematocysts isolated from C. nozakii (VEC) or N. nomurai (VEN) using a modi fied version of the technique described by Carrette and Seymour (2004). In brief, nematocysts were resuspended using Hanks’ balance salt solution(HBSS, NaCl 137 mmol/L, KCl 5.6 mmol/L, CaCl21.26 mmol/L, MgSO40.81 mmol/L, Na2HPO40.38 mmol/L, KH2PO40.44 mmol/L and NaHCO34.2 mmol/L, the pH of HBSS was adjusted to 7.4 with 0.1 mol/L HCl and NaOH) containing 1 mmol/L of phenylmethylsulfonyl fluoride (PMSF). Before extraction, 100 μL of each nematocyst resuspension was examined using microscope respectively to check the discharge condition of the nematocysts,and the number of the nematocysts prepared for venom extraction was obtained by microscope count method. One milliliter of the nematocyst resuspension solution was placed into screw-top vials with 2.5 g of glass beads (0.5 mm in diameter),and the mixture was shaken five times in a mini bead beater (Mini-Beadbeater-16, Biospec Products,Bartlesville, USA) at 3 450 r/min for 20-s intervals with intermittent cooling on ice. After ensuring the absolute discharge of the nematocysts in resuspension microscopically, the venom was then centrifuged at 6 824× g at 4°C for 20 min and the supernatant used for the present study. The protein concentration of the venom extracts was determined following the method of Bradford (1976) and compared with bovine serum albumin (BSA) protein concentration standards, and the venoms were then used in the hemolytic and cytotoxicity assays based on their protein concentrations.

    2.3 Hemolytic activity assay

    The hemolytic activity of the venoms was tested using erythrocytes from chicken, pigeon, and sheep,which could easily be obtained from the local market.In brief, fresh blood samples were mixed with 1 mg/mL of heparin sodium as an anticoagulant at a 1:10 volume immediately after blood collection.Whole blood (60 mL) was centrifuged at 684× g for 10 min at 4°C, the supernatant was removed gently,and the erythrocytes were washed three times with HBSS. The erythrocytes were then resuspended in the same buffer to make a 1% (v/v) solution for the following hemolysis assay.

    Various volumes of jelly fish venoms (0.1, 0.3, 0.5,0.7 and 0.9 mL) were added to 0.5 mL of the erythrocyte suspension obtained from the three species (chicken, pigeon, and sheep). The venomerythrocyte mixtures were adjusted using HBSS to a final volume of 1.5 mL and final protein concentrations of venoms (16, 49, 82, 115, and 147 μg/mL,respectively) were checked as mentioned above. All the mixtures were incubated at 37°C for 30 min. Then,1 mL of each mixture was diluted with 4 mL of HBSS and centrifuged at 1 000× g for 5 min at 4°C. The supernatants were transferred to 96-well microplates and the absorbance at 410 nm was determined using a visible spectrophotometer (7230G, Shanghai, China)to measure the extent of erythrocyte lysis. The half maximal effective concentration (EC50) values of each concentration of jelly fish venom protein were calculated to evaluate the hemolytic activity of the jelly fish venoms.

    2.4 Cell culture and treatment

    The A431 human epidermal carcinoma cell line was purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM Medium (Gibco, USA) with 4 mmol/L of L-glutamine, 100 μg/mL of streptomycin and 100 U/mL of penicillin, plus 15% fetal calf serum(FCS, TBD, China), and incubated at 37°C in a humidi fied 5% CO2atmosphere.

    2.5 Cytotoxicity assay

    Cytotoxicity was assessed by measuring the mitochondrial dehydrogenase activity, using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Brie fly, cells were plated on 96-well plates at a density of 4×103cells/well and cultured under normal conditions for 24 h. Venoms were diluted with HBSS to the same concentrations described above (16, 49, 82, 115, and 147 μg/mL),and added to pre-cultured cells, respectively. All incubations with various protein concentrations were then stopped at 24, 48, 72 and 96 h. The control group was treated simultaneously with HBSS only.

    After incubation, the treated cells were observed and photographed using an inverted phase-contrast microscope (Nikon, Japan). Two hundred microliters of MTT solution (2.5 mg/mL) was added to each well and the plates were incubated for another 4 h at 37°C.The medium was then removed and replaced by 200 μL of dimethyl sulfoxide (DMSO) to solubilize the formazan crystal generated in the culture plates.The absorbance at 490 nm was read using a spectrophotometric microplate reader (MultiSkan FC,Thermo Scienti fic, USA) and the cell survival ratio(%) was calculated using Eq.1:

    The half maximal inhibitory concentration (IC50)values were calculated to evaluate the cytotoxic activity of jelly fish venoms.

    2.6 Statistical analysis

    All data were expressed as means±SD of three parallel measurements. The SPSS16.0 software was used for statistical analysis, and statistically signi ficant differences between groups were determined by oneway ANOVA. For further comparison between two groups, the Student-Newman-Keuls (SNK) method was used for equal variance, whereas the Games-Howell test was used for unequal variances.Differences were considered signi ficant at P <0.05.EC50and IC50values were calculated using a probit regression model.

    3 RESULT

    3.1 Observations of nematocysts

    As shown in Table 1, the density of nematocysts isolated from C. nozakii or N. nomurai was calculated after observations under an inverted microscope.There were fewer nematocysts per unit area from tentacles of N. nomurai than from tentacles of C. nozakii. Nematocysts isolated from tentacles of N. nomurai were predominant spheroidal, whereas those from C. nozakii were oval (Fig.1).

    Fig.1 The microscopic structure of nematocysts isolated from N. nomurai (left) and C. nozakii (right)

    3.2 The total protein content of nematocyst venoms

    The protein concentration per nematocyst was calculated based on the protein concentration of the venoms and nematocyst suspension density. As shown in Fig.2, the protein concentration of VEN was higher than VEC at the same nematocyst density. Therefore,the protein concentration per nematocyst from N. nomurai was higher than that of C. nozakii.Interestingly, Fig.2 (left side) shows lower concentrations of protein per nematocyst with the increasing of nematocyst densities. That was probably due to the partly discharged nematocysts in resuspension during the process of venom extraction, and protein in supernatant might induce an overestimate of protein concentration per nematocyst at lower density.

    3.3 Hemolysis

    The hemolytic activities of venoms were assessed using erythrocyte suspensions from chicken, pigeon,and sheep. Both VEC and VEN showed strong hemolytic activities. After incubation with either VEN or VEC, all chicken, pigeon, and sheep erythrocytes showed obvious hemolytic reaction including morphological changes and obvious lysis (Fig.3)

    Fig.2 Comparison of the protein concentration of nematocysts (left) and venoms (right) from C. nozakii (white bars) and N.nomurai (black bars)

    Table 1 The density of nematocysts in tentacles of N. nomurai and C. nozakii

    Fig.3 Morphological changes of chicken, pigeon, and sheep erythrocytes before (upper) and after (lower) incubation with 147 μg/mL protein concentration of jelly fish venoms for 30 min

    Fig.4 Hemolytic effects of VEN (white bars) and VEC (black bars) on erythrocytes from chickens after 30- (a) or 60- (b) min incubation ( n=3, P <0.05)

    Venom extracts were applied at concentrations of 16, 49, 82, 115 and 147 μg/mL. Absorbance analysis with a one-way ANOVA showed that the time- and concentration-dependent hemolytic effects were in fluenced signi ficantly by the protein concentration and incubation time ( P <0.01). As shown in Figs.4–6,treatment with 16 μg/mL of VEC for 30 min resulted in hemolysis ratios in chicken, pigeon, and sheep erythrocytes of 22.4%, 19.8% and 24.7%, respectively,whereas the hemolysis ratios of the erythrocytes treated with the same concentration of VEN were 13.8%, 12.5% and 23.8%, respectively. With an extended treatment time of 60 min, the hemolysis ratios of chicken, pigeon, and sheep erythrocytes treated with VEC increased to 26.2%, 27.6% and 33.5%, respectively, whereas the hemolysis ratios of the erythrocytes treated with VEN increased to 21.6%,24.5% and 27.1%, respectively. The hemolytic effects of both jelly fish venoms at other protein concentrations showed similar differences, and all erythrocytes showed complete lysis after incubation with 147 μg/mL of venom (Figs.4–6).

    Fig.5 Hemolytic effects of VEN (white bars) and VEC (black bars) on erythrocytes from pigeons after 30- (a) or 60- (b) min incubation ( n=3, P <0.05)

    Fig.6 Hemolytic effects of VEN (white bars) and VEC (black bars) on erythrocytes from sheep after 30- (a) or 60- (b) min incubation ( n=3, P <0.05)

    Fig.7 EC 50 of VEC (black bars) and VEN (white bars) on erythrocytes from chicken, pigeon, and sheep at different exposure times

    Fig.8 Number of nematocysts from N. nomurai (white bars)and C. nozakii (black bars) required to meet the EC 50 level on chicken, pigeon, and sheep erythrocytes over 30- and 60-min exposure times

    The EC50of both VEC and VEN on erythrocytes from the three species reduced over time ( P <0.05).Comparison of the EC50results indicated that sheep nematocysts were the most sensitive to the hemolytic effects of both jelly fish venoms, and VEC had a stronger hemolytic activity than VEN at the same protein concentrations (Fig.7). In addition, to enable the hemolytic activity to reach the EC50level, the number of nematocysts and tentacle area required from C. nozakii and N. nomurai were calculated according to the nematocyst densities listed above. Comparisons showed that similar numbers of nematocysts from the two species were needed to reach the EC50level in chicken, pigeon, and sheep erythrocytes for either the 30- or 60-min exposure time (Fig.8). Given that the number of nematocysts per unit area in tentacles of N. nomurai was lower than in C. nozakii, the tentacle area of N. nomurai corresponding to the EC50levels on erythrocytes from the three species was larger compared with C. nozakii (Fig.9).

    Fig.9 Tentacle areas of N. nomurai (white bars) and C. nozakii (black bars) required to meet the EC 50 level on chicken, pigeon, and sheep erythrocytes over 30- and 60-min exposure times

    3.4 Cytotoxicty

    Morphological changes in A431 cells following their exposure to the jelly fish venoms were observed under a microscope. The cells of the untreated control group grew well and their nuclei were round and clear. Cells treated with either VEC or VEN were misshapen and some were broken. Nuclei were marginalized in some cells (Fig.10). The cytotoxic activities of the venoms were evaluated using the MTT assay, and the results showed a dose-dependent inhibition of either VEN or VEC on cell viability with increasing protein concentrations (Fig.10). After incubation with VEC and VEN for 24 h at a concentration of 16 μg/mL, the survival rates of A431 cells were 64% and 80.2%, respectively. The survival rates at 24 h reduced to 8.1% and 9.5% when the protein concentrations of venoms increased to 147 μg/mL. Meanwhile, the cytotoxic activities of both venoms were signi ficantly related to incubation time ( P <0.05). After incubation for 96 h with VEC and VEN, the survival rates of A431 cells at a concentration of 16 μg/mL reduced to 49.7% and 52.8%, respectively. The survival rates of cells incubated with 147 μg/mL of venom reduced to 5.5%and 5.6%, respectively. Lower values of IC50for VEC at different incubation times (Fig.11) compared with VEN were also recorded (Figs.12, 13).

    Fig.10 Morphological changes in A431 cells following 24-h incubation with different concentrations of jelly fish venom

    Fig.11 Comparison of the cytotoxic effects of VEC (black bars) and VEN (white bars) on the viability of A431 cells following their incubation with venom for 24, 48, 72, and 96 h

    Fig.12 Number of nematocysts of N. nomurai (white bars)and C. nozakii (black bars) needed to meet the IC 50 cytotoxic level on A431 human epidermal carcinoma cells over different incubation times

    Fig.13 Tentacle area of N. nomurai (white bars) and C.nozakii (black bars) needed to meet the IC 50 cytotoxic level on A431 human epidermal carcinoma cells over different incubation times

    4 DISCUSSION

    The jelly fish C. nozakii is widely distributed along the coast of China and blooms have been reported in the northern East China Sea, Yellow Sea, and Bohai Sea (Dong et al., 2010). It can ruin fishing nets, prey on juvenile fish, crabs and mollusks, and produce toxins that are poisonous to humans and marine animals (Zhou and Huang, 1956; Peng and Zhang,1999; Zhong et al., 2004; Dong et al., 2006). In humans, the venom of C. nozakii can produce a burning feeling that develops into severe pain,although no deaths have been attributed to this species(Tibballs, 2006; Helmholz et al., 2007). N. nomurai is also a dominant species throughout seas along East China, including Bohai Sea, Yellow Sea, and northern East China Sea (Kawahara et al., 2006). Blooms of N.nomurai were reported in the northern part of the East China Sea, Yellow Sea, and Liaodong Bay in 2003,2005, and 2007, respectively (Cheng et al., 2004;Ding and Cheng, 2005). This species was responsible for most of the severe or fatal cases of jelly fish stings in Chinese seas (Zhang et al., 1993; Xu et al., 2007;Jiang et al., 2008). Given that hemolytic proteins in nematocyst venoms are key molecules determining the toxic effects of jelly fish stings, including erythema and edema (Li et al., 2013), the hemolytic activity of venoms extracted from jelly fish tentacle nematocysts has been widely reported against erythrocytes from different species (Bloom et al., 2001; Torres et al.,2001; Nagai et al., 2002; Tibballs, 2006). The sensitivity with which erythrocytes re flect these conditions varies from species to species (Table 2).Kang et al. (2009) assessed the hemolytic activity of N. nomurai on cat, dog, human, rabbit, and rat erythrocytes and showed a concentration-dependent activity of venom extracts starting at 10 μg/mL of protein equivalents; dog erythrocytes were the most sensitive (EC50=151 μg/mL, 30 min). The hemolytic activity of extracts from C. nozakii was also documented on human and chicken erythrocytes(Helmholz et al., 2007; Feng et al., 2009, 2010). This study compared the hemolytic activities of venoms extracted from nematocysts of C. nozakii and N. nomurai on erythrocyte suspensions from chickens,pigeons, and sheep. Sheep erythrocytes were the most sensitive to the hemolytic effects of these two jelly fish venoms, with EC50values of VEN and VEC of 69.69 and 63.62 μg/mL over a 30-min exposure. In addition,the venom from C. nozakii showed a stronger hemolytic activity than that from N. nomurai on all three erythrocyte suspensions at the same protein concentration. Based on the same relative amount of each jelly fish nematocyst needed to reach the EC50level on erythrocytes, the hemolytic activities per nematocyst from N. nomurai and C. nozakii were similar. However, the tentacle area of N. nomurai corresponding to the EC50level was larger than that of C. nozakii. Thus, the hemolytic activity per unit area of tentacle from C. nozakii was stronger than that of N. nomurai.

    Fig.14 The IC 50 of N. nomurai (white bars) and C. nozakii(black bars) nematocyst venoms on A9, A673, and A431 cells following 24–96-h exposure

    There are two general mechanisms to explain this hemolytic activity. The first is an enzymatic mechanism,whereby cytolytic components bind preferentially to membrane glycolipids or glycoproteins (Burnett and Calton, 1987). The other is a stoichiometric mechanism,whereby toxin molecules bind to, and insert in, the plasma membrane followed by oligomerization to form transmembrane pores (Bhakdi and Tranum-Jensen, 1988). Many studies have indicated that jelly fish venoms form pore-like structures in target membranes, causing rapid cell lysis (Rottini et al.,1995; Edwards et al., 2002), and that the pore formation only occurs at the site of envenoming, possibly serving to aid the entry of other venom components into the host (Bailey et al., 2005). Thus, the hemolytic activity is likely to occur in combination with the envenomation of the jelly fish venom. However, it is not fully understood how the venoms of C. nozakii and N. nomurai elicit their hemolytic potencies in different species, and so requires further investigation.

    Cytotoxicity is also a common bioactivity of jelly fish venoms (Table 3). Li et al. (2012) assessed the cytotoxicity of C. nozakii venom on Bel-7402 and SMMC-7721 human hepatoma cells and H630 human colon cancer cells. H630 cells were most sensitive to the venom (IC50=15.9, 8.8, and 5.1 μg/mL after incubation for 12, 24, and 48 h, respectively) followed by Bel-7402 (17.9 μg/mL) and SMMC-7721(24.3 μg/mL). C2C12 (muscle myoblast) and H9C2(heart myoblast) cell lines were used to assess the cytotoxicity of crude venom from N. nomurai, which showed high cytotoxic effects against H9C2 heart myoblasts (IC50=2 μg/mL; Kang et al., 2009). Lee et al.(2011) assessed the cytotoxicity of venom from four scyphozoan jelly fish ( Nemopilema nomurai, Rhopilema esculenta, C. nozakii, and Aurelia aurita) on NIH 3T3 mouse fibroblast cells, and reported a cytotoxic potency scale of C. nozakii > N. nomurai > A. aurita >R. esculenta. We had previously compared the cytotoxicity of venoms from C. nozakii and N. nomurai on A9 (mouse subcutaneous connective tissue) and A673 (human rhabdomyosarcoma) (Xu et al., 2014).The results from both our studies demonstrated that venom from C. nozakii nematocysts was more cytotoxically potent than that from N. nomurai nematocysts at the same protein concentration. Further calculations also indicated the stronger cytotoxic activities per nematocyst and per unit area of tentacle from C. nozakii compared with N. nomurai. In addition,a comparison of IC50values of C. nozakii and N. nomurai nematocyst venoms on A9, A673, and A431 cells showed that A431 cells were most sensitive to the cytotoxic effects of both jelly fish venoms (Fig.14).

    In conclusion, our results showed that both C. nozakii and N. nomurai jelly fish venoms have strong hemolytic and cytotoxic activities, which might be attributed to the protein content of the venoms, with venoms from C. nozakii showing stronger toxic activities than those from N. nomurai. Sheep erythrocytes were the most sensitive to the hemolytic effects of both jelly fish venoms, and the EC50values of venoms from N. nomurai and C. nozakii were 69.69 and 63.62 μg/mL following 30-min exposure, respectively. The IC50values of venoms from N. nomurai and C. nozakii were 68.6 and 40.9 μg/mL, respectively, following 24-h incubation with A431 human epidermal carcinoma cells. Given that jelly fish nematocysts contain at least one toxic component, further biochemical investigations are required to characterize the different components of jelly fish nematocyst extracts and to clarify their mechanism of action.

    5 CONCLUSION

    Comparative study on the toxic activities of crude venoms from C. nozakii and N. nomurai nematocysts shows that nematocyst venom from C. nozakii had stronger hemolytic and cytotoxic activities than that from N. nomurai. We concluded that C. nozakii might be more harmful to the health of humans than be N.nomurai, and, thus, people are advised to pay more attention on the risk of sting by C. nozakii.

    男人和女人高潮做爰伦理| 99re6热这里在线精品视频| 国产免费一级a男人的天堂| 国产真实伦视频高清在线观看| 亚洲国产欧美人成| av女优亚洲男人天堂| 直男gayav资源| 欧美日本视频| 国产午夜精品久久久久久一区二区三区| 黑人高潮一二区| 嫩草影院新地址| 最近2019中文字幕mv第一页| 欧美一级a爱片免费观看看| 三级男女做爰猛烈吃奶摸视频| 亚洲天堂av无毛| 日韩av免费高清视频| 午夜福利视频1000在线观看| 老女人水多毛片| 成人亚洲精品一区在线观看 | 成人亚洲精品一区在线观看 | 色吧在线观看| 欧美xxxx黑人xx丫x性爽| 极品教师在线视频| 亚洲在久久综合| 国产极品天堂在线| 久久人人爽人人爽人人片va| 在现免费观看毛片| 成人一区二区视频在线观看| 身体一侧抽搐| 一本—道久久a久久精品蜜桃钙片 精品乱码久久久久久99久播 | 久热这里只有精品99| 永久网站在线| 日韩av免费高清视频| 2021少妇久久久久久久久久久| 伊人久久国产一区二区| 国产熟女欧美一区二区| 一本—道久久a久久精品蜜桃钙片 精品乱码久久久久久99久播 | 国产成人一区二区在线| 精品人妻视频免费看| 在线观看国产h片| 下体分泌物呈黄色| 赤兔流量卡办理| 成人毛片a级毛片在线播放| 中国三级夫妇交换| av国产久精品久网站免费入址| 国产爽快片一区二区三区| 伊人久久精品亚洲午夜| 亚洲熟女精品中文字幕| av国产精品久久久久影院| 国产精品一二三区在线看| 免费少妇av软件| 亚洲电影在线观看av| 久久精品久久久久久噜噜老黄| 听说在线观看完整版免费高清| 51国产日韩欧美| 国产色婷婷99| 久久久亚洲精品成人影院| 爱豆传媒免费全集在线观看| 黄色视频在线播放观看不卡| 亚洲丝袜综合中文字幕| 日产精品乱码卡一卡2卡三| 日本猛色少妇xxxxx猛交久久| 热99国产精品久久久久久7| 老师上课跳d突然被开到最大视频| 黄色日韩在线| 亚洲精品aⅴ在线观看| 久久久久久伊人网av| 一个人观看的视频www高清免费观看| 女的被弄到高潮叫床怎么办| 三级男女做爰猛烈吃奶摸视频| 亚洲伊人久久精品综合| 亚洲va在线va天堂va国产| 尤物成人国产欧美一区二区三区| 亚洲欧美精品自产自拍| 各种免费的搞黄视频| 婷婷色综合www| 国产大屁股一区二区在线视频| 欧美日韩综合久久久久久| 一级毛片 在线播放| 国产在视频线精品| 亚洲丝袜综合中文字幕| 国产一区二区三区av在线| 黄色怎么调成土黄色| 国产一级毛片在线| .国产精品久久| 精品久久久久久电影网| 成年人午夜在线观看视频| 成人综合一区亚洲| 国产精品国产三级专区第一集| 噜噜噜噜噜久久久久久91| 麻豆乱淫一区二区| 久久精品国产亚洲av涩爱| 日本午夜av视频| 国精品久久久久久国模美| 中文字幕亚洲精品专区| 肉色欧美久久久久久久蜜桃 | 看黄色毛片网站| 男人舔奶头视频| 青春草亚洲视频在线观看| 女人被狂操c到高潮| 欧美+日韩+精品| 亚洲天堂av无毛| 色播亚洲综合网| 国产在线一区二区三区精| 色网站视频免费| 爱豆传媒免费全集在线观看| 国产视频内射| 人人妻人人澡人人爽人人夜夜| 亚洲怡红院男人天堂| 插阴视频在线观看视频| 国产伦精品一区二区三区四那| 久久久亚洲精品成人影院| 中国国产av一级| 婷婷色av中文字幕| 女人久久www免费人成看片| 久久99热这里只有精品18| 卡戴珊不雅视频在线播放| 国产乱人偷精品视频| 最近中文字幕2019免费版| 亚洲av免费在线观看| 精品一区二区三区视频在线| 色5月婷婷丁香| 亚洲精品日韩av片在线观看| 18禁裸乳无遮挡免费网站照片| 午夜精品一区二区三区免费看| 国产乱人视频| 91在线精品国自产拍蜜月| 精品99又大又爽又粗少妇毛片| 看十八女毛片水多多多| 亚洲国产欧美在线一区| 少妇人妻精品综合一区二区| 91狼人影院| 少妇被粗大猛烈的视频| 日本欧美国产在线视频| 91狼人影院| 日韩精品有码人妻一区| 中文乱码字字幕精品一区二区三区| 午夜免费观看性视频| 亚洲一区二区三区欧美精品 | 最近中文字幕2019免费版| 中文精品一卡2卡3卡4更新| 欧美xxxx性猛交bbbb| 亚洲无线观看免费| 免费av毛片视频| 毛片女人毛片| 麻豆成人av视频| 肉色欧美久久久久久久蜜桃 | 91精品一卡2卡3卡4卡| 自拍偷自拍亚洲精品老妇| 亚洲av电影在线观看一区二区三区 | 少妇 在线观看| 久久久久久久亚洲中文字幕| 国产老妇女一区| 一本色道久久久久久精品综合| 国产老妇伦熟女老妇高清| 成人午夜精彩视频在线观看| 美女主播在线视频| 国产老妇伦熟女老妇高清| 日韩在线高清观看一区二区三区| 一区二区av电影网| 亚洲欧美一区二区三区国产| 亚洲av二区三区四区| 国产一区亚洲一区在线观看| 午夜激情福利司机影院| av福利片在线观看| 久久影院123| 99热这里只有精品一区| 熟女电影av网| 深爱激情五月婷婷| 国产 一区 欧美 日韩| 精品人妻偷拍中文字幕| 日日摸夜夜添夜夜爱| 高清在线视频一区二区三区| 国产爱豆传媒在线观看| 久久精品久久精品一区二区三区| 午夜精品一区二区三区免费看| 亚洲精品久久久久久婷婷小说| 久久精品久久精品一区二区三区| 可以在线观看毛片的网站| 久久99蜜桃精品久久| 一二三四中文在线观看免费高清| 久久精品熟女亚洲av麻豆精品| av在线观看视频网站免费| 69av精品久久久久久| 丝袜喷水一区| 日本色播在线视频| 中文乱码字字幕精品一区二区三区| 91狼人影院| 亚洲综合色惰| 啦啦啦啦在线视频资源| 肉色欧美久久久久久久蜜桃 | 一个人看的www免费观看视频| 各种免费的搞黄视频| 麻豆乱淫一区二区| 制服丝袜香蕉在线| 卡戴珊不雅视频在线播放| 成人综合一区亚洲| 一区二区av电影网| 性色av一级| 老司机影院毛片| 男女边吃奶边做爰视频| 伊人久久国产一区二区| 日韩av在线免费看完整版不卡| 国产高清有码在线观看视频| 午夜免费男女啪啪视频观看| 丰满人妻一区二区三区视频av| 哪个播放器可以免费观看大片| 天天躁日日操中文字幕| 欧美潮喷喷水| 久久久成人免费电影| 亚洲欧美一区二区三区国产| 在线天堂最新版资源| 国产精品爽爽va在线观看网站| 中文字幕亚洲精品专区| 国产亚洲5aaaaa淫片| 亚洲高清免费不卡视频| 成人午夜精彩视频在线观看| 国产成人91sexporn| 国产精品一区www在线观看| 国产探花极品一区二区| 欧美另类一区| 乱码一卡2卡4卡精品| 亚洲av国产av综合av卡| 亚洲欧美清纯卡通| 日本熟妇午夜| av一本久久久久| 亚洲精品一区蜜桃| 国产精品无大码| 久久国内精品自在自线图片| 伦理电影大哥的女人| 麻豆国产97在线/欧美| 如何舔出高潮| 蜜臀久久99精品久久宅男| 精品久久久精品久久久| 久久久欧美国产精品| 一区二区av电影网| 水蜜桃什么品种好| 国产午夜福利久久久久久| 99九九线精品视频在线观看视频| 精品一区二区三卡| 精品久久久久久电影网| 国产精品蜜桃在线观看| 蜜臀久久99精品久久宅男| 性色avwww在线观看| 人人妻人人爽人人添夜夜欢视频 | 寂寞人妻少妇视频99o| 亚洲精品久久午夜乱码| 亚洲人成网站在线观看播放| 免费电影在线观看免费观看| 国产爽快片一区二区三区| 日韩av在线免费看完整版不卡| 国模一区二区三区四区视频| 久久久久国产网址| 国产精品女同一区二区软件| 日韩一区二区视频免费看| 日韩精品有码人妻一区| 婷婷色av中文字幕| 日产精品乱码卡一卡2卡三| 高清欧美精品videossex| 高清视频免费观看一区二区| 久久久久久国产a免费观看| 亚洲图色成人| 精华霜和精华液先用哪个| 日韩亚洲欧美综合| 99精国产麻豆久久婷婷| 91在线精品国自产拍蜜月| 日韩一区二区视频免费看| 亚洲综合色惰| 欧美成人午夜免费资源| 国产精品无大码| 简卡轻食公司| 久久女婷五月综合色啪小说 | 欧美97在线视频| 亚洲美女视频黄频| 色视频www国产| 大话2 男鬼变身卡| 春色校园在线视频观看| 女人久久www免费人成看片| 国产高清国产精品国产三级 | 午夜福利在线观看免费完整高清在| 亚洲,一卡二卡三卡| 欧美高清成人免费视频www| 色吧在线观看| av黄色大香蕉| 搡女人真爽免费视频火全软件| 青春草国产在线视频| 国产午夜福利久久久久久| 卡戴珊不雅视频在线播放| 久久亚洲国产成人精品v| 又粗又硬又长又爽又黄的视频| 国产91av在线免费观看| 精品久久久久久久久亚洲| 韩国av在线不卡| 寂寞人妻少妇视频99o| 少妇猛男粗大的猛烈进出视频 | 欧美xxxx黑人xx丫x性爽| 欧美人与善性xxx| 日韩av免费高清视频| 中文字幕人妻熟人妻熟丝袜美| av国产免费在线观看| 亚洲成人久久爱视频| 欧美日韩综合久久久久久| 国产av国产精品国产| 亚洲无线观看免费| 国产精品精品国产色婷婷| 成年女人在线观看亚洲视频 | 精品久久久久久久人妻蜜臀av| 成人国产av品久久久| 亚洲精品日韩在线中文字幕| 久久久久精品性色| 午夜福利网站1000一区二区三区| 亚洲欧美清纯卡通| 日本免费在线观看一区| 丰满少妇做爰视频| 欧美最新免费一区二区三区| 国产免费视频播放在线视频| 五月开心婷婷网| 色视频在线一区二区三区| 国产精品国产av在线观看| 亚洲av福利一区| 久久精品国产鲁丝片午夜精品| 婷婷色综合大香蕉| 嘟嘟电影网在线观看| 国产亚洲5aaaaa淫片| 亚洲成人久久爱视频| 日本黄大片高清| av国产久精品久网站免费入址| 国产成人精品福利久久| 嫩草影院新地址| 人妻一区二区av| 欧美另类一区| 人人妻人人看人人澡| 一级a做视频免费观看| 丝袜喷水一区| 亚洲av免费在线观看| 新久久久久国产一级毛片| 久久精品国产自在天天线| 成人二区视频| 精华霜和精华液先用哪个| 性色av一级| 蜜臀久久99精品久久宅男| 日韩 亚洲 欧美在线| 成人毛片60女人毛片免费| 国语对白做爰xxxⅹ性视频网站| h日本视频在线播放| 少妇裸体淫交视频免费看高清| 精品久久久久久久人妻蜜臀av| 日韩av免费高清视频| 夜夜爽夜夜爽视频| 国产av不卡久久| 观看免费一级毛片| 国产欧美另类精品又又久久亚洲欧美| 天堂网av新在线| 色吧在线观看| 成人美女网站在线观看视频| 国产精品人妻久久久影院| 国产色爽女视频免费观看| 亚洲欧洲国产日韩| 国内精品宾馆在线| 在线观看人妻少妇| 亚洲av男天堂| 国产亚洲av片在线观看秒播厂| 日本与韩国留学比较| 久久精品夜色国产| freevideosex欧美| 成人鲁丝片一二三区免费| 亚洲真实伦在线观看| 51国产日韩欧美| 中文字幕av成人在线电影| 中文乱码字字幕精品一区二区三区| 永久网站在线| 下体分泌物呈黄色| 亚洲一区二区三区欧美精品 | 少妇人妻一区二区三区视频| 国产一级毛片在线| 又大又黄又爽视频免费| 日本三级黄在线观看| 少妇人妻 视频| 国产欧美日韩一区二区三区在线 | 最近最新中文字幕大全电影3| 久久久久久伊人网av| 国产爽快片一区二区三区| 精品国产乱码久久久久久小说| 一级av片app| 97人妻精品一区二区三区麻豆| 啦啦啦啦在线视频资源| 黄片wwwwww| 国产精品久久久久久久久免| 成人国产麻豆网| 日本三级黄在线观看| 久久99热这里只有精品18| 亚洲精品一区蜜桃| 亚洲av免费在线观看| 国产日韩欧美在线精品| 日韩三级伦理在线观看| 美女被艹到高潮喷水动态| 一边亲一边摸免费视频| 小蜜桃在线观看免费完整版高清| 欧美性感艳星| 国产毛片a区久久久久| 啦啦啦中文免费视频观看日本| 免费大片黄手机在线观看| 免费观看性生交大片5| 高清视频免费观看一区二区| 69av精品久久久久久| h日本视频在线播放| av国产久精品久网站免费入址| 精品国产一区二区三区久久久樱花 | 久久久久久国产a免费观看| 国产在线男女| 免费大片18禁| 成年版毛片免费区| 丰满少妇做爰视频| 人妻夜夜爽99麻豆av| 水蜜桃什么品种好| av播播在线观看一区| 久热久热在线精品观看| 少妇人妻 视频| 国产在线一区二区三区精| www.色视频.com| 老师上课跳d突然被开到最大视频| 免费不卡的大黄色大毛片视频在线观看| 欧美一级a爱片免费观看看| 日韩av免费高清视频| 波多野结衣巨乳人妻| av在线天堂中文字幕| 精品少妇黑人巨大在线播放| 国产免费视频播放在线视频| 三级男女做爰猛烈吃奶摸视频| 春色校园在线视频观看| 国产乱人视频| 婷婷色av中文字幕| 精品一区二区免费观看| 男女那种视频在线观看| 成人特级av手机在线观看| 尾随美女入室| 毛片一级片免费看久久久久| 全区人妻精品视频| 亚洲国产色片| av播播在线观看一区| 亚洲天堂av无毛| 免费在线观看成人毛片| 免费观看a级毛片全部| 日本三级黄在线观看| 97超视频在线观看视频| av专区在线播放| av在线天堂中文字幕| 久久久久国产网址| 亚洲欧美清纯卡通| 亚洲精品乱码久久久久久按摩| 三级国产精品片| 精品久久久久久久久亚洲| 高清在线视频一区二区三区| 国产成人福利小说| 特级一级黄色大片| 国产免费一区二区三区四区乱码| 国产女主播在线喷水免费视频网站| 一级黄片播放器| 97人妻精品一区二区三区麻豆| 国产在线一区二区三区精| 精品午夜福利在线看| 久久久精品免费免费高清| 在线观看美女被高潮喷水网站| 欧美激情久久久久久爽电影| 高清欧美精品videossex| 国产永久视频网站| 日本黄色片子视频| 成人国产麻豆网| 亚洲av成人精品一区久久| 色哟哟·www| 久久亚洲国产成人精品v| 日日摸夜夜添夜夜爱| 夜夜看夜夜爽夜夜摸| 国产黄色视频一区二区在线观看| 五月伊人婷婷丁香| 亚洲av成人精品一区久久| 综合色av麻豆| 黄色配什么色好看| 高清欧美精品videossex| 男女啪啪激烈高潮av片| 国产一级毛片在线| 亚洲av免费在线观看| 免费看不卡的av| 91在线精品国自产拍蜜月| 九九在线视频观看精品| 狂野欧美激情性bbbbbb| 亚洲成人中文字幕在线播放| 乱码一卡2卡4卡精品| 欧美另类一区| 白带黄色成豆腐渣| 男人和女人高潮做爰伦理| av在线观看视频网站免费| 熟妇人妻不卡中文字幕| 亚洲国产欧美在线一区| 久久久久久久久久成人| 日韩免费高清中文字幕av| 男人舔奶头视频| 狠狠精品人妻久久久久久综合| av又黄又爽大尺度在线免费看| 在线播放无遮挡| 毛片一级片免费看久久久久| 久久久久久九九精品二区国产| 亚洲成人一二三区av| 国产成人免费观看mmmm| 欧美变态另类bdsm刘玥| 一二三四中文在线观看免费高清| 成人毛片60女人毛片免费| 免费看av在线观看网站| 丰满人妻一区二区三区视频av| 久久久欧美国产精品| 91精品伊人久久大香线蕉| 国产女主播在线喷水免费视频网站| 超碰97精品在线观看| 少妇人妻久久综合中文| 精品久久久久久久人妻蜜臀av| 一区二区三区四区激情视频| 99视频精品全部免费 在线| 狂野欧美白嫩少妇大欣赏| 26uuu在线亚洲综合色| 日韩视频在线欧美| 综合色丁香网| 国产精品久久久久久久电影| 99热全是精品| 亚洲av男天堂| 中国美白少妇内射xxxbb| 成人无遮挡网站| 亚洲激情五月婷婷啪啪| 亚洲人成网站在线播| 欧美精品国产亚洲| 日韩,欧美,国产一区二区三区| 国产亚洲av嫩草精品影院| 2021天堂中文幕一二区在线观| 日日啪夜夜爽| 青春草亚洲视频在线观看| 国产老妇女一区| 人妻系列 视频| 少妇人妻久久综合中文| 赤兔流量卡办理| 国产高清有码在线观看视频| 国产女主播在线喷水免费视频网站| 国产精品久久久久久久久免| 又粗又硬又长又爽又黄的视频| 欧美+日韩+精品| 国产亚洲精品久久久com| 国产午夜精品久久久久久一区二区三区| 国产亚洲av片在线观看秒播厂| 成人漫画全彩无遮挡| 精品国产一区二区三区久久久樱花 | 亚洲高清免费不卡视频| 亚洲精品视频女| 国产乱人偷精品视频| 我要看日韩黄色一级片| 精品国产露脸久久av麻豆| 国产精品一二三区在线看| 国产伦精品一区二区三区四那| 女人十人毛片免费观看3o分钟| 在线看a的网站| 亚洲精品456在线播放app| 黑人高潮一二区| 免费看光身美女| 国产淫片久久久久久久久| 精华霜和精华液先用哪个| 精品视频人人做人人爽| 黄色怎么调成土黄色| 成人毛片60女人毛片免费| 久久久久久久国产电影| 久久久精品94久久精品| 人人妻人人澡人人爽人人夜夜| 一级黄片播放器| 色吧在线观看| 91狼人影院| 免费观看的影片在线观看| 亚洲自拍偷在线| 成人综合一区亚洲| 久久韩国三级中文字幕| 久久久午夜欧美精品| 观看免费一级毛片| 在线观看美女被高潮喷水网站| 亚洲第一区二区三区不卡| 嘟嘟电影网在线观看| 有码 亚洲区| 久久久久久久久久久免费av| 少妇熟女欧美另类| 爱豆传媒免费全集在线观看| 搞女人的毛片| 99久国产av精品国产电影| 欧美日韩精品成人综合77777| 人妻 亚洲 视频| 97热精品久久久久久| 91aial.com中文字幕在线观看| 亚洲三级黄色毛片| 嫩草影院入口| 中文欧美无线码| 久久精品熟女亚洲av麻豆精品| 精品久久久久久久末码| 国产精品成人在线| 大又大粗又爽又黄少妇毛片口| 69av精品久久久久久| 神马国产精品三级电影在线观看| 男女那种视频在线观看| 女人十人毛片免费观看3o分钟| 亚洲精品亚洲一区二区| tube8黄色片| 亚洲,一卡二卡三卡| 国产精品一区二区性色av| 欧美成人午夜免费资源| 一级二级三级毛片免费看| 三级国产精品欧美在线观看| 国产久久久一区二区三区| 久久综合国产亚洲精品| 91久久精品电影网| av在线app专区| 午夜免费鲁丝| 男女啪啪激烈高潮av片| 男女那种视频在线观看|