Neuroprotective effect of Qinggan Lishui formula on retinal ganglion cell apoptosis in a microbead-induced rat chronic glaucoma model

INTRODUCTION

Glaucoma is an optic nerve degenerative disease,which is predicted to affect＞80 million people worldwide by 2020,with approximately 10%being bilaterally blind.1,2

Decreasing intraocular pressure(IOP)has long been regarded as the only effective method for treatment of glaucoma.3In addition,current available pharmacological treatments include cholinergic drugs,β-adrenergic agonists,β-adrenergic receptor blockers,α-adrenoreceptor agonists,carbonic anhydrase inhibitors,and prostaglandin derivatives.4,5However,all of these drugs exhibit local or systemic side effects,such as conjunctival hyperemia,uveitis,macular edema,eye irritation,dry eye,headache,and apnea.6-8The pathophysiological hallmark of glaucoma is progressive apoptotic death of retinal ganglion cells(RGCs).9-12Thus,there is an urgent need for complementary medicines that facilitate the survival of RGCs during treatment of glaucoma.

数学与图论跟其他有着完善理论和问题解决办法的体系不同，其分支不同，问题涉及比较广泛，同时有着多样的问题解决方法，一般情况下，一类问题往往存在一种解法，然而不同的解法间缺少一些相关的联系。有一句老话说道，“工欲善其事，必先利其器”，数学竞赛中要使用图论问题，就得先对图论进行了解、探索，其中要了解图论存在哪些问题以及常见的处理问题的方法，再具体进行运用。而在图论问题中，主要研究其组合最值以及存在性两个问题。

The decoction from Qinggan Lishui formula(QF),consisting of four Chinese herbs,was first used by Dr.Zhou Jusheng,who is an experienced expert in Traditional Chinese Medicine(TCM)at Longhua Hospital of Shanghai.Our previous studies have shown that QF may improve vision acuity in glaucoma patients when co-administered with eye drops that reduce intraocular pressure(IOP),and that the effective rate of IOP reduction measured by Goldmann applanation tonometer in the QF in combination with eye drops group was significantly higher than that in the eye drop alone group.13In addition,QF treatment may attenuate RGC loss in an experimental glaucoma rat model.14In the present study,we explored possible mechanisms for the protective effect of QF on RGCs in a rat model of chronic intraocular hypertension(COH).

MATERIALS AND METHODS

Preparation of QF extract

QF consists of four herbs,as shown in Table 1,which are listed in the database of www.theplantlist.org.The herbs were mixed in a proportion of 4∶5∶5∶4(Table 1).Ten-fold ddH2O was added and boiled twice,for 1 h each time.The raw drug liquid was concentrated to a final concentration of 1 g dried crude herb per mL of liquid,to produce QF extract,which was filtered and stored at－80℃until used.

Table 1 Herbals contained within the Qinggan Lishui formula

Ratio Common name Prunella Pueraria Plantago Wolfberry Chinese name Xiakucao Gegen Cheqianzi Gouqizi 4 5 5 4 Botanical name Prunella vulgaris L Pueraria candollei benth Plantago seetzenii decne Lycium barbarum L

Animals and rat COH model

Experimental procedures in the present study were performed in compliance with National Institutes of Health(NIH)guidelines for the Care and Use of Laboratory Animals and the rules of Shanghai University of Traditional Chinese Medicine regarding ethical use of animals (Certificate No. 2015-05-2132). Male Sprague-Dawley rats,weighing 150-250 g,were purchased from the Shanghai SLAC Laboratory Animal Co.,Ltd.,(Shanghai,China),and housed on a 12 h light/dark schedule.During this study,all possible efforts were made to minimize the number of rats used and their suffering.

REFERENCES

QF administration

From D1 onwards,COH rats were given QFviaintragastric administration(gavage),once daily at a dose of 6.2 g/kg,until D28.COH rats that received an equal volume of physiological solution(saline)served as control animals.

Retrograde labeling of RGCs

RGCs were retrogradely labeled following a previously described procedure.17,18Briefly,after rats were deeply anesthetized with 10%chloral hydrate(4 mL/kg),1%cholera toxin B subunit(CTB,List Biological Laboratories,Campbell,CA,USA)was injected into the bilateral superior colliculus(2 μL each site,6.0 mm posterior and 2.0 mm lateral to the bregma,5 mm deep from the cortical surface).Rats were killed after 5 d of survival and an anti-CTB antibody was used to detect CTB labeled RGCs.

CTB immunohistochemistry experiments were performed according to previously described procedures.18,19Animals were anesthetized and killed by decapitation.

Eyes were removed rapidly and fixed with 4%paraformaldehyde(PFA)in 0.1 M phosphate buffer(PB)for 2 h at room temperature,then dehydrated with graded sucrose solutions(2 h each in 10%and 20%,then overnight in 30%).The eyecups were embedded in OCT compound(Tissue Tek,Torrance,CA,USA)and then stored at−80℃ until further use.

Whole flat-mounted retinal CTB immunohistochemical staining was performed.18,19Retinas were mounted on the slide and rinsed with 0.01 M PBS,then blocked for 1 h in 5%normal donkey serum(NDS,Sigma,St.Louis,MO,USA)in PBS with 0.3%Triton X-100 at room temperature.Retinas were then incubated with a primary antibody,polyclonal goat anti-CTB(1∶4000 dilution)for 5 consecutive days at 4℃.Sites that bound to the primary antibody were visualized by incubating with Cy3-conjugated donkey anti-goat IgG(1∶400 dilution;Sigma-Aldrich St Louis,MO,USA)for 1 d.CTB-immunoreactivity was visualized using a confocal laser scanning microscope,through a 20×objective(Fluoview 1000,Olympus,Monolith,Tokyo,Japan).

另外，厌氧池中的丝状菌对活性污泥絮粒的聚集有阻碍作用，从而引起系统活性污泥流失，影响厌氧池反硝化处理效果，同时丝状菌的聚集作用一定程度上增加了系统剩余污泥量。为了避免厌氧池内污泥菌群比例失调，提高厌氧池反硝化处理效果，进入厌氧池的循环管线设置位于池中部，并在末端安装带有均匀分布出水孔的管排，如图2所示。厌氧池内厌氧分解及反硝化脱氮均可充分进行，起到调节CNP比例的作用。

Assay for apoptosis of RGCs

To detect cell apoptosis,the terminal dUTP nick end labeling(TUNEL)assay was performed on whole flat-mounted retinas,18-20using the DeadEnd Fluorometric TUNEL System G3250 kit(Promega,Madison,WI,USA)and following manufacturer's instructions.TUNEL signals were visualized using a confocal laser scanning microscope,through a 10×objective(Fluoview 1000,Olympus,Monolith,Tokyo,Japan).The retina was mounted with the ganglion cell layer(GCL)upturned,and serial deep scans were performed only in the GCL,as determined by 4',6-diamidino-2-phenylindole(DAPI)staining.All TUNEL-positive signals that merged appropriately with DAPI were counted in each retina.

Quantification of RGCs and TUNEL positive signals

CTB-labeled RGCs and TUNEL-positive signals in whole flat retinas were counted following a previously described procedure.18Briefly,two representative regions were selected,which are designated as central and peripheral regions throughout this report.An area located at＜1.0 mm(commonly between 200 and 830 μm)distant from the optic nerve head(ONH)was defined as the central region,whereas an area located 3.0-3.63 mm distant from the ONH was defined as the peripheral region.To quantify the number of CTB-or TUNEL-positive cells,two fields,one each from the central and peripheral regions,were selected at each of four angles of the retina(0°,90°,180°,and 270°),for a total of eight fields;the numbers of CTB-or TUNEL-positive cells were counted in each of these fields.Each field area was 0.397 mm2(630 μm × 630 μm).Allimages were acquired and exported using FV10-ASW Viewer 1.7 software(Olympus,Tokyo,Japan),then assembled using Adobe Photoshop.

Statistical analysis

Data analysis was performed using SPSS 18.0(SPSS Inc.,Chicago,IL,USA);data are presented as mean±standard error of mean.throughout the text.Statistical analysis was performed using one-way analysis of variance with Bonferroni post test or Kruskal-Wallis test.A value ofP＜0.05 was considered to be significant.

作为单案例研究，本文有一定的局限性，即难以建构社会组织合法化路径和策略的一般性理论。参与社区治理的社会组织类型多样，组织的合法性禀赋也有较大差别。但是，对草根社会组织LL合法化过程的分析，展现了社会组织在合法化过程中如何选择、操控和创造环境的策略。这对于各类社会组织有普遍性的借鉴意义，可以帮助社会组织科学制定参与社区治理的微观策略。同时，这些结论也有助于政府在宏观层面制定更加科学的政策，培育社会组织能力，推动社会组织参与社区治理，从整体上促进基层治理创新和社会领域健康发展。社会组织合法化路径和策略的分类型研究，对社会组织自身特征与合法化路径的结合研究，应当成为未来研究的重点。

RESULTS

Steady elevation of IOPs in rat glaucoma model

Changes in IOP were monitored in microbead-injected and contralateral eyes of injected rats.Mean IOP significantly increased to(16.0±0.5)mm Hg on D1 following microbead injection[compared with(9.6±0.1)mm Hg before injection and(9.7±0.1)mm Hg in contralateral eyes](n=32,P＜0.001),and consistently maintained this higher level[ranging from(16.0±0.1)to(19.1±1.1)mm Hg,n=8-24,allP＜0.001 vs control]through D28(Figure 1A).In addition,beginning on D1,the mean IOP in microbead-injected eyes was also higher than mean IOP in saline-injected eyes[ranging from(9.3±0.4)to(10.4±0.2)mm Hg,n=24,allP＜0.001](Figure 1B).These results suggest that microbead injection can successfully induce a rat COH model.However,in QF-treated COH rats,mean IOP significantly declined,beginning on D14[(15.7±0.7)mm Hg,n=24,P＜0.001]and continuing through D28[(13.3±1.1)mm Hg,n=8,P＜0.05](Figure 1C,D),compared with eyes in the COH-only group,suggesting that QF administration may reduce IOP in the rat COH model.

QF administration reduces RGC loss in COH rats

We first assessed survival of RGCs in our rat glaucoma model using the CTB retrograde-labeling technique.Figure 2 shows representative micrographs of a saline-injected retina(control)(Aa1),and COH retinas on D7,D14,D21,and D28 after microbead injection(Aa2-Aa5);these reveal a progressive decrease in the number of CTB-labeled RGCs at different times post-injection.CTB-labeled RGCs and their axons were clearly seen in all of the micrographs.Specifically,no obvious changes were observed in the number of RGCs in COH eyes on D7,compared with control eyes(Figure 2A).The average number of CTB-labeled RGCs within eight fields from COH eyes on D7 was 699±17/field(n=4,P＞0.05),which was similar to the number in control eyes(713±18/field,n=3).However,the number of CTB-positive RGCs in COH eyes was significantly reduced on D14(605±21/field,n=4,P＜0.01vscontrol)and the reduction became more conspicuous by D21(485±25/field,n=4,P＜0.001vscontrol)and D28(326±26/field,n=4,P＜0.001vscontrol).In contrast,QF administration significantly attenuated RGC loss in COH eyes.Even though no obvious changes in number of RGCs were observed on D7 after QF administration(711±20/field,n=4,P＞0.05vscontrol)(Figure 2G),the average numbers of CTB-labeled RGCs decreased to 669±18/field(n=4),563±21/field(n=4),and 437±25/field(n=4)on D14,D21,and D28 respectively;all of these measurements were significantly higher than the corresponding values in COH rats that did not receive QF,at the same time points post-injection(allP＜0.05)(Figure 2H-J).

Figure 1 Intraocular pressure(IOP)changes of both eyes in a rat model of glaucoma

A:bar chart showing average IOP changes in microbead-injected eyes and corresponding contralateral eyes at different times post-injection.B:bar chart showing average IOP changes in saline-injected eyes and corresponding contralateral eyes at different times post-injection.C:bar chart showing average IOP changes in microbead-injected eyes and corresponding contralateral eyes at different times post-injection.Rats received Qinggan Lishui formulaviaintragastric administration(gavage)from D1 onwards,following microbead injection,once daily at a dose of 6.2 g/kg until D28.D:changes in IOP as a function of time under different conditions.All data are presented as mean±standard error of mean.aP＜0.05 andbP＜0.001vsleft eyes in control rats;cP＜0.05 anddP＜0.001vscontralateral right eyes at the same time point.

Figure 2 Qinggan Lishui formula(QF)administration attenuates the decrease in number of Cholera toxin B subunit(CTB)retrogradely labeled retinal ganglion cells(RGCs)in a rat model of glaucoma

A:representative images of whole flat-mounted retinas from saline-injected eyes(A),and microbead-injected eyes on D7,D14,D21,and D28,respectively(B-E).Note that the number of CTB-labeled RGCs progressively decreased from D7 onwards.B:representative images of retinas from untreated eyes(F),and QF-treated microbead-injected eyes at the same post-injection time points(G-J)as shown in Line A.Note that QF administration significantly attenuated the elevated intraocular pressure-induced decrease in the number of CTB-labeled RGCs.

RGC apoptosis is reduced by QF administration in COH rats

We then explored possible mechanisms contributing to the protective effect of QF administration on RGCs in COH retinas.TUNEL staining was performed to detect apoptosis of RGCs in COH retinas.As illustrated in Figure 3,sparse TUNEL-positive signals were detected in flat-mounted COH retinas on D7 after microbead injection(Figure 3 B1-B3).TUNEL-positive signals increased on D14,with an average number of 87±7/field(n=4,P＜0.05vscontrol)(Figure 3 C1-C3).Numerous TUNEL-positive signals were observed on D21(Figure 3 D1-D3)and D28(Figure 3 E1-E3)in COH retinas;the average numbers of TUNEL-positive cells increased to 224±22/field(n=4,P＜0.001vscontrol)on D21 and 608±31/field(n=4,P＜0.001vscontrol)on D28,respectively.In QF-treated retinas,elevation of IOP-induced RGC apoptosis was significantly attenuated.Beginning on D14,QF administration reduced the average numbers of TUNEL-positive cells to 56±6/field(n=4,P＜0.05vsCOH retinas)on D14,161±14/field(n=4,P＜0.05vsCOH retinas)on D21,and 468±29/field(n=4,P＜0.05vsCOH retinas)on D28,respectively.Figure 3F shows representative micrographs of a QF-treated COH retina on D28(F1-F3).

DISCUSSION

In the present study,we found that QF decoction may moderately reduce IOP and significantly attenuate RGC loss in the microbead injection-induced rat COH model,and that these effects are mediated by inhibition of RGC apoptosis.Thus,reduction of RGC apoptosis may contribute to the protective effect of QF against vision loss and RGC damage caused by glaucoma.

Clinical evidence suggests a potential relationship between elevated IOP and glaucoma.In previous studies,a variety of experimental glaucoma animal models have been established to study the mechanisms of RGC death;the methods to produce these animal models include ligation of episcleral veins,injection of polystyrene microbeads,and laser photocoagulation of the trabecular meshwork.15,16,18,19,21-24In the present study,we injected superparamagnetic iron oxide microbeads into the anterior chamber of rat eyes.Following injection,the microbeads were distributed around the iridocorne-al angle,resulting in blockade of the uveoscleral and trabecular outflow pathways.15,16Subsequently,IOP was elevated at D1 and remained elevated for at least 4 weeks,with the IOP of injected eyes consistently approximating 19.1 mm Hg;this was significantly higher than the IOP of control eyes(approximately 9.6 mm Hg).The difference in IOPs was quite similar to prior reports that utilized the polystyrene microbead-injected mouse model.15,16Our results suggest that superparamagnetic iron oxide microbead injection can induce sustained elevation of IOP.

Figure 3 Qinggan Lishui formula(QF)administration reduces the number ofTerminal dUTP nick end labeling(TUNEL)-positive retinal ganglion cells(RGCs)in a rat model of glaucoma

A;B;C;D;E:representative confocal microphotographs of TUNEL signals(green)in whole flat-mounted retinas from control eyes(a1)and microbead-injected eyes on D7(B1),D14(C1),D21(D1),and D28(E1),respectively.Images in A2,B2,C2,D2,and E2 show counterstaining with 4',6-diamidino-2-phenylindole(DAPI,blue).Images in a3,b3,c3,d3,and e3 are merged images of corresponding TUNEL and DAPI images.Note that sparse TUNEL-positive signals were detected on D14,and that these signals increased in density on D21 and D28 after injection.F:representative confocal microphotographs ofTUNEL signals(green)in whole flat-mounted retinas from QF-treated microbead-injected eyes on D28(F1).Image in F2 shows counterstaining with DAPI(blue).Image in F3 is a merged image of the corresponding TUNEL and DAPI images.Note that QF administration significantly reduced the number of TUNEL-positive signals,compared with microbead injection alone(E1).Inset images in the left corner are respective enlarged images of corresponding fields(squares),showing thatTUNEL signals clearly overlapped with DAPI staining.

In our glaucomatous rat model,RGC loss was first detected on D14,followed by severe loss on D21 and D28(Figure 2);these findings are consistent with previous studies of the COH rat model.18Importantly,we found that QF administration significantly attenuated the reduction in number of CTB-labeled RGCs in COH rats.One possible mechanism may be that QF moderately reduced IOP in these rats(Figure 1).As QF consists of four Chinese herbs,the protective effect of QF on RGCs in COH retina may result from a complex set of mechanisms.Previous studies have shown that these four herbs may provide neuronal protective effects in cases of various retinal diseases and injuries,viaincreases in antioxidant ability and anti-inflammatory effects,as well as decreases in oxidative injury.25-32Specifically,Xiakucao(Spica Prunella Vulgaris)and its ethanol extracts could inhibit production of nitric oxide and may be useful in treatment of inflammation and eye pain.25,26Puerariafuran,extracted from the roots of Gegen(Radix Puerariae lobatae),could significantly increase glutathione/oxidized glutathione ratio,superoxide dismutase(SOD),and catalase activity in a model of xylose-induced lens opacity.27Puerarin also effectively improved axoplasmic transport in the optic nerve and ameliorated disc area microcirculation in a rabbit model of COH,and it provided protective effects for optic nerves in a rat model of retinal ischemia-reperfusion injury—these effects were mediated by increased SOD activity and decreased malonyldialdehyde content.28,29Plantago has been shown to inhibit lens epithelial cell apoptosis induced by H2O2,through increased antioxidative ability and decreased oxidative injury.30-32Furthermore,wolfberry and its extracts could protect RGCs against injuries in many retinal diseases,including experimental glaucoma,ischemia-reperfusion injury,and partial optic nerve transaction.33-39In conclusion,even though QF decoction may provide protective effect for RGCs in glaucomatous retinas,it may not be sufficient as a monotherapy for treatment of glaucoma,and should be administered alongside other therapies.

The rat COH model was reproduced following a previously described procedure,with some modifications.15,16Briefly,after rats were anesthetized by intraperitoneal injection of 10%chloral hydrate(4 mL/kg),5 μL of microbeads(superpara-magnetic iron oxide,approximately 10 μm in size,50 mg/mL)(Bangs Laboratories,Indianapoli,IN,USA)were injected into the anterior chamber of each rat's left eye,under an OPMI VISU 140 microscope(Carl Zeiss,Jena,Germany).After the injection,the microbeads were drawn away from the injection site and distributed around the iridocorneal angle,using a small handheld magnet(0.45 Tesla),to decrease the outflow of aqueous humorviathe trabecular meshwork.Sham injection,following a similar procedure(except for the injection of 0.9%NaCl solution,rather than microbeads),was conventionally performed on the eyes of the remaining rats.IOP was measured using a handheld digital tonometer(Tonolab,TioLat,Finland)under general and local anesthesia.The average value of five consecutive measurements with a deviation less than 5%was accepted.All measurements were performed in the morning to avoid possible circadian interference.IOPs of both eyes were measured before injection(baseline),immediately after injection(D0),1 d after injection(D1),3 and 7 d after injection(D3 and D7),and weekly afterwards(D14,D21,and D28)through 28 d post-injection.

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周恩来和郭沫若此时正在武汉组织纪念抗战一周年群众歌咏大会，得知桂涛声和冼星海创作了这首《在太行山上》，便前往冼星海住所先睹为快。

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1950—1970年，中南佛州工程共实施了20年。该项工程主要包括五项内容：一是河道整治，二是胡佛坝的加固与加高，三是修建了多个地表存水区，四是大范围修建防护堤，五是建设排水渠网及入海处防止海水入侵结构物。共修建了1 700 km运河、约1 200 km堤坝、200座控水建筑物、15座主要泵站等。

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11 Resnikoff S,Pascolini D,Etya'ale D,et al.Global data on visual impairment in the year 2002.Bull World Health Organ 2004;82(1):844-851.

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13 Dong ZG,Zhang YJ,Liu XQ.Clinical study on the effect of Qinggan Lishui Mingmu method for visual functional protection on primary open angle glaucoma with the syndrome of liver depression andQistagnation.J Tradi Chin Ophthalmol 2013;5(7):336-339.

14 Wang LL,Zhang YJ.Protective effects of Qinggan Lishuiformula on intraocular pressure.J Tradi Chin Med 2015;5(5):5-12.

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23 Lu W,Hu H,Sévigny J,et al.Rat,mouse,and primate models of chronic glaucoma show sustained elevation of extracellular ATP and altered purinergic signaling in the posterior eye.Invest Ophthalmol Vis Sci 2015;56(5):3075-3083.

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7 Li J,Lin X,Yu M.Meta-analysis of randomized controlled trials comparing latanoprost with other glaucoma medications in chronic angle-closure glaucoma.Eur J Ophthalmology 2015;25(1):18-26.

倡导民生水利的发展理念，就要求人们不能就水利看水利，就工程论工程，而要跳出水利看水利，始终站在民生的角度审视和发展水利，把惠泽民生作为水利工作的价值追求，贯穿于水利工作的各个领域，自始至终，矢志不渝。

20 Gavrieli Y,Sherman Y,Bensasson SA,et al.Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol 1992;119(6):493-501.

渠道策略：应结合产品设计，因人而异，因地制宜开展多种营销。例如，应积极与大中型事业企业联系,鼓励企业组织员工到景区来进行郊游、户外拓展训练、徒步活动等,并给予折扣。

一是以学讲话促进秋收生产。垦区各地牢记总书记努力当好维护国家粮食安全“压舱石”，把“中国粮食，中国饭碗”抓在手上、记在心中的嘱托，以讲话精神为动力，全力加强秋收工作，确保颗粒归仓、保质保量。集团成立了以总经理为组长的秋收工作领导小组，逐级分片包点，明确任务、落实责任，科学指导、靠前指挥、昼夜抢收。截至10月25日，垦区4300余万亩耕地秋收工作全面告捷，预计全年总产430亿斤以上，创历史新高。截至11月5日，垦区高标准完成4300余万亩耕地“黑色越冬”工作。水田备苗床土400多万立方米，秋做床1.3亿多平方米，旱田秋起垄1300多万亩，为2019年农业生产奠定了良好基础。

22 Ji M,Miao Y,Dong LD,et al.Group I mGluR-mediated inhibition of Kir channels contributes to retinal muller cell gliosis in a rat chronic ocular hypertension model.J Neurosci 2012;32(6):12744-12755.

17 Zhao WJ,Zhang M,Miao Y,et al.Melatonin potentiates glycine currents through a PLC/PKC signaling pathway in ratretinal ganglion cells.JPhysiol2010;588(2):2605-2619.

24 Yun H,Lathrop KL,Yang E,et al.A laser-induced mouse model with long-term intraocular pressure elevation.PLoS One 2014;9(9):e107446.

25 Hwang YJ,Lee EJ,Kim HR,et al.In vitroantioxidant and anticancer effects of solvent fractions from Prunella vulgaris var.lilacina.BMC Complement Altern Med 2013;13(1):310.

26 Kong FY,Ng DC,Chan CH,et al.Parental use of the term"HotQi"to describe symptoms in their children in Hong Kong:a cross sectional survey"HotQi"in children.J Ethnobiol Ethnomed 2006;2(7):2.

27 Kim,NH,Kim YS,Lee YM,et al.Inhibition of aldose reductase and xylose-induced lens opacity by puerariafuran from the roots of pueraria lobata.Biol Pharm Bull 2010;33(9):1605-1609.

28 Xuan B,Zhou YH,Yang RL,et al.Improvement of ocular blood flow and retinal functions with puerarin analogs.J Ocul Pharmacol Ther 1999;15(3):207-216.

29 He L,Wang XS,Han LY.Protective effect of puerarin on optic nervers after experimental retinal is chemia-reper fusion injury.RecAdvin Ophthalmol2006;12(2):916-918.

30 Wang Y,QI MX,Huang XR,Wang ZY.Experimental studay on semen plantaginis inhibiting LEC apoptosis induced by oxidative damage of lens.Xian Dai Zhen Duan Yu Zhi Liao 2003;4(4):199-202.

通过以上分析，我们可以推断，留学生的语言能力与其语用语言能力有一定的关系，但与其社交语用能力之间关系不大。留学生的社交语用能力比其语用语言能力发展得要慢。这就要求我们在教学中加强对学生社交语用能力的培养。

2 Quigley HA,Broman AT.The number of people with glaucoma worldwide in 2010 and 2020.Br J Ophthalmol 2006;90(3):262-267.

32 Huang XR,Qi MX,Yan J,et al.Signal transduction mechanism of the protective effect of semen plantaginis on oxidative damage of lens epithelial cells.Chim J Clin Pharmacol Therapeutics 2008;7(7):768-771.

33 Chan HC,Chang RC,Chiu WH,et al.Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma.Exp Neurol 2007;203(1):269-273.

34 Mi XS,Feng Q,Lo AC,et al.Protection of retinal ganglion cells and retinal vasculature by Lycium barbarum polysaccharides in a mouse model of acute ocular hypertension.PLoS One 2012;7(10):e45469.

受到手机网络影响，学生群体求知方式发生改变，很多知识点网上搜索一下就有，学生不引起重视。老师布置作业，学生多数依葫芦画瓢，并没真正掌握写作要领，往往在日后工作中碰壁。

35 Chiu K,Chan HC,Yeung SC,et al.Modulation of microglia by Wolf-berry on the survival of retinal ganglion cells in a rat ocular hypertension model.J Ocul Biol Dis Infor 2009;2(2):47-56.

36 Chiu K,Zhou Y,Yeung SC,et al.Up-regulation of crystallins is involved in the neuroprotective effect of wolfberry on survival of retinal ganglion cells in rat ocular hypertension model.J Cell Biochem 2010;110(2):311-320.

37 Li SY,Yang D,Yeung CM,et al.Lycium barbarum polysaccha rides reduce neuronal damage,blood-retinal barrier disruption and oxidative stress in retinal ischemia reperfusion injury.PLoS One 2011;6(1):e16380.

38 He M,Pan H,Chang RC,et al.Activation of the Nrf2/HO-1 antioxidant pathway contributes to the protective effects of Lycium barbarum polysaccharides in the rodent retina after ischemia-reperfusion induced damage.PLoS One 2014;9(1):e84800.

39 Chu PH,Li HY,Chin MP,et al.Effect of Lycium barbarum(wolfberry)polysaccharides on preserving retinal function after partial optic nerve transection.PLoS One 2013;8(12):e81339.

CRISPR/Cas系统对病毒DNA清除能力为病毒的治疗提供了新思路。利用CRISPR/Cas系统能够有效安全地将HIV病毒从体外培养的人T细胞的DNA中清除。我国军事医学科学院放射与辐射医学院研究所、第四军医大学西京医院、日本京都大学等处的研究人员研究靶向乙肝表面抗原(HBsAg)编码区的CRISPR/Cas9系统，在体外培养的肝细胞和活小鼠体内的效果表明CRISPR/Cas9可在体内和体外抑制HBV复制和表达，可能是治疗HBV感染的一种新策略[13]。