Biochemical Thiab Histopathological Teb Ntawm Biomphalaria Alexandrina Rau RIPEX (cog Loj hlob Regulator)Ⅰ

Abstract

Keeb kwm

Cog kev loj hlob regulators yog dav siv nyob rau hauv kev ua liaj ua teb rau kev loj hlob thiab ripening ntawm cov nroj tsuag, tab sis lawv ua rau muaj kev phom sij rau cov dej ecosystem. Txoj kev tshawb fawb tam sim no tau soj ntsuam cov txiaj ntsig ntawm sublethal raug rau RIPEX 48 feem pua ​​​​EC concentrations (8 thiab 16 µL/L) ntawm oxidative stress parameters, poj niam txiv neej cov tshuaj hormones, lub cev tsis muaj zog enzymes, txawv-tial hemocyte suav, thiab histopathology ntawm digestive glands thiab ovotestis hauv Biomphalaria alexandrina qwj.

Nyem rau cistanche herba rau testosterone

Cov txiaj ntsig

RIPEX raug tshwm sim ua rau tag nrho nce hauv Superoxide dismutase thiab Glutathione-S-Transferase cov dej num hauv B. Alexandrina. Txawm li cas los xij, RIPEX exposure heev inhibits SOD kev ua haujlwm hauv cov qwj. Malondialdehyde kev ua ub no qhia tau tias muaj kev nce ntxiv hauv B. Alexandrina raug rau ob qho tib si ntau tom qab txhua lub sijhawm raug. RIPEX kuj ua rau muaj kev nce ntxiv hauv testosterone hauv cov qwj raug rau 16 µL / L, nws tau ua, txawm li cas los xij, txo qis cov tshuaj hormones hauv cov qwj raug rau 8 µL / L ntawm 7 hnub. Hais txog estradiol, muaj qhov nce ntxiv tom qab 3 hnub ntawm kev kis mus rau 16 µl / L thiab 7 hnub ntawm kev raug rau 8 µL / L. RIPEX exposure kuj nce cov dej num ntawm Myeloperoxidase thiab Adenosine deaminase enzymes nyob rau hauv lub digestive qog ntawm qwj. Nws nce tag nrho cov hemocyte suav ntawm cov qwj raug pom nrog rau cov granulocytes. Snail digestive caj pas thiab ovotestis pom cov kab mob hloov pauv tom qab 7 hnub ntawm RIPEX raug.

Cov lus xaus

Cov kev tshawb pom no qhia tias RIPEX yog tshuaj lom rau B. Alexandrina thiab tias cov qwj no tuaj yeem siv los ua bioindicator rau ib puag ncig kev sib kis nrog cov nroj tsuag kev loj hlob.

Ntsiab lus

Biomphalaria alexandrina, RIPEX, Testosterone, Estradiol, Myeloperoxidase, Adenosine deaminase, Histopathology

1 Tom qab

Agrochemical pollution ntawm cov dej saum npoo av yog ib qho kev txhawj xeeb thoob ntiaj teb ib puag ncig, tshwj xeeb tshaj yog nyob rau hauv cov cheeb tsam uas kev ua liaj ua teb tau nthuav dav thiab nthuav dav. Ntau yam tshuaj hluavtaws, xws li tshuaj tua kab lossis tshuaj chiv, yog siv los hloov cov qoob loo kom muaj nuj nqis thiab zoo kom tau raws li cov neeg siv khoom xav tau [7, 40]. Cov neeg txhawb nqa kev loj hlob yog cov tshuaj agrochemicals hauv ntau lub tebchaws [18].

Thaum cov tshuaj no ncaj qha nqus los yog noj nrog cov zaub tsis huv, lawv ua rau tib neeg noj qab haus huv [39]. Cov tshuaj tua kab organophosphorus, ethephon (2- chloroethyl phosphonic acid) yog ib qho tshuaj ethylene-tso hluavtaws uas feem ntau siv los ua cov nroj tsuag kev loj hlob (PGR) los txhawb kev induction fower induction, txiv hmab txiv ntoo ripening, thiab lwm yam physiological tshua. Nws raug pom zoo siv rau ntau yam khoom noj, pub, tsis yog khoom noj, tsev cog khoom cog, thiab cov nroj tsuag ornamental cog rau sab nraum zoov [3, 50]. Kev ntsuam xyuas ntawm ethephon toxicity qhia tau hais tias ethephon induced morphometric malformations ntawm lub thib ob kav hlau txais xov, sensory bristles, tail spine, thiab rostrum ntawm Daphnia magna uas ua rau poob ntawm kev ua haujlwm thiab kev tuag [54].

Abd-El Azeem [1] tau sau tseg qhov txo qis ntawm kev loj hlob, DNA puas, thiab apoptotic tshwm sim hauv plab hnyuv hlwb ntawm slug Deroceras reticulatum tom qab raug ethephon. Ntau tus kws sau ntawv pom zoo rau toxicity ntawm Ethephon rau nas thiab noog [2, 17, 55]. Tsis tas li ntawd, ethephon raug ua rau genotoxicity, oxidative kev nyuaj siab, thiab kev ua me nyuam toxicity hauv nas [16]. Teratogenicity thiab gonadal system dysfunction hauv nas kuj tau txuas nrog ethephon raug [2, 16]. Ethephon kuj tau pom tias yog immunotoxic, muaj mutagenic zoo [26], nce oxidative kev nyuaj siab, thiab qis deoxyribonucleic thiab Ribonucleic acids concentrations [4].

Ethephon nyob rau hauv lub cev dej yuav ua rau muaj kev phom sij rau cov tsiaj hauv dej; Txawm li cas los xij, cov ntaub ntawv ntawm nws qhov cuam tshuam rau cov kab mob hauv dej tsis tshua muaj. Ntau hom tsiaj hauv dej tau siv los txheeb xyuas cov tshuaj hormones cog qoob loo hauv dej. Txawm li cas los xij, qee cov kab mob no nyuaj rau kev tswj hwm thiab tswj xyuas hauv chav kuaj, ua rau lawv nyuaj rau siv hauv kev tshuaj ntsuam ecotoxicological [54]. Cov dej mollusks, nrog rau cov qwj, tsis ntev los no tau txais kev saib xyuas raws li cov kab mob hauv lub cev vim qhov xav tau los tsim cov qauv invertebrate rau kev saib xyuas ib puag ncig ntawm cov kab mob hauv dej [21, 45]. Snails yog pawg mollusk ntau tshaj plaws hauv cov dej hauv dej. Snails nthuav tawm qhov kev siv tsawg dua rau cov vertebrates siab dua li bioindicators vim tias lawv tuaj yeem siv ntau yam kev sim yam tsis tau nrhiav kev pom zoo rau kev coj ncaj ncees, thiab lawv qhov kev tau txais thiab kev saib xyuas yog pheej yig [13].

Hauv qhov kev hwm no, Biomphalaria alexandrina snail tau dav siv los ua ib qho kev qhia txog kab mob ntawm cov dej tsis huv nrog tshuaj tua kab [29, 30]. Tshaj li nws lub peev xwm los ua tus ntsuas bioindicator, tus qwj no ua tus tswv tsev nruab nrab rau kev sib kis ntawm Schistosoma mansoni [35]. Yog li ntawd, agrochemical concentrations nyob rau hauv ib puag ncig tuaj yeem cuam tshuam kev kis tus kab mob schistosomiasis los ntawm kev cuam tshuam ncaj qha thiab tsis ncaj rau ntawm tus tswv tsev nruab nrab thiab cov kab mob cab [27]. Kev tshawb nrhiav tam sim no tsom rau kev tshawb xyuas qhov cuam tshuam ntawm sublethal raug rau cov nroj tsuag kev loj hlob regulator, RIPEX 48 feem pua ​​EC (lub npe lag luam ntawm ethephon, 2-chloroethyl phosphonic acid) ntawm ntau yam biochemical, immunological, thiab histological yam ntawm B. alexandrina snails raws li bioindicator kab mob.

2 Txoj kev

2.1 Test lub cev

Biomphalaria alexandrina snails tau sau los ntawm Lake Manzalah nyob hauv El-Matareya, Dakahlia Governorate, Tim lyiv teb chaws (31 degree 11′ N 32 degree 2′ E) thiab khaws cia rau hauv cov dej zoo aerated ntawm qhov kub thiab txias ntawm 20 degree. Lawv tau noj tshiab lettuce nplooj txhua hnub. Tom qab txij nkawm rau 4 lub lis piam nyob rau hauv lub chaw kuaj mob, noj qab nyob zoo snails (10-12 mm nyob rau hauv lub plhaub taub) tau siv nyob rau hauv lub tom ntej thwmsim.

2.2 Kev sim cov khoom siv thiab raug mob

Cov nroj tsuag kev loj hlob regulator, RIPEX (daim ntawv tshuaj muaj 48 feem pua ​​​​ethephon [2- chloroethyl phosphonic acid (C2H6ClO3P)] thiab 52 feem pua ​​inert cov khoom xyaw) tau muas los ntawm Chema Industries (New Nubaria nroog, Behira, Egypt; daim ntawv tso cai No. . 1840). Ib series ntawm concentrations tau npaj. Tsob ntoo replicas nrog kaum snails txhua tus tau raug. Tom qab 1- hnub dhau los, cov qwj tuag raug suav, thiab tshem tawm thiab LC50 thiab LC90 raug suav los ntawm cov ntaub ntawv txheeb cais ntawm social sciences (SPSS; IBM Corp. Armonk, NY, USA).

2.3 Kev sim tsim

Ob lub sublethal concentrations sawv cev rau LC10 thiab LC20 tau siv (8 thiab 16 µL/L). 90 snails tau muab faib ua peb pawg, txhua pab pawg nrog peb replicates ( kaum snails / replicate): pawg tswj, pawg 8 µL/L-exposed, thiab pawg 16 µL/L-exposed. Ntawm 1-, 3-, thiab 7-hnub ncua sij hawm, oxidative stress biomarkers thiab tiv thaiv kab mob enzymes tau raug soj ntsuam nyob rau hauv lub caj pas digestive. Cov tshuaj hormones steroid tau soj ntsuam hauv Ovotestis. Kev txheeb xyuas qhov sib txawv ntawm hemocytes, thiab histology ntawm lub plab zom mov thiab ovotestis tau soj ntsuam. Rau kev tshawb nrhiav biochemical, 0.5 g ntawm cov ntaub so ntswg (cov qog digestive lossis ovotestis) los ntawm txhua pab pawg yog homogenized hauv 0.5 mL phosphate tsis (pH 6.5) siv lub khob homogenizer thiab centrifuged ntawm 10, 000 × g, 4 degree rau 10 min. . Cov txiaj ntsig supernatants tau siv rau kev soj ntsuam tom ntej.

2.4 Kev txiav txim siab ntawm oxidative stress markers

2.4.1 Superoxide dismutase (SOD, EC 1.15.1.1)

Kev ua SOD tau txiav txim siab siv EnzyChrom™ Superoxide Dismutase Assay Kit (Cat. No: ESOD{{0}}}; BioAssay Systems, Hayward, CA, USA) raws li cov chaw tsim khoom cov lus qhia. Cov ntaub so ntswg homogenate siv txias lysis buffer (50 mM potassium phosphate, 0.1 mM EDTA, thiab 0.5 feem pua ​​Triton X-100) tau centrifuged ntawm 12,000g rau 5 min ntawm 4 degree , thiab tus supernatant tau sau rau kev txiav txim siab ntawm SOD [43]. Kev ua haujlwm ntawm SOD tau ntsuas ntawm qhov muag pom qhov muag (OD) ntawm 440 nm.

2.4.2 Glutathione-S-Transferase (GST, EC 2.5.1.18)

Tis enzyme catalyzes GSH conjugation nrog {{0}}chloro-2, 4-dinitrobenzene (CDNB). Nws yog ntsuas spectrophotometrically ntawm 340 nm. Cov tshuaj sib tov muaj cov poov tshuaj phosphate tsis 0.1 M, pH 6.5, GSH yaj hauv cov poov tshuaj phosphate tsis (140 mg / 100 mL), thiab CDNB 4 mg / ml hauv ethyl cawv. Ib chav tsev ntawm enzyme kev ua haujlwm yog txhais tau tias yog tus nqi ntawm cov enzyme uas catalyzes tsim ntawm 1 μmole ntawm S-conjugate ib feeb [11].

2.4.3 Malondialdehyde (MDA)

Malondialdehyde nyob rau hauv tag nrho homogenates tau txiav txim siab raws li thiobarbituric acid reactive tshuaj (TBARS) raws li tus qauv txheej txheem ntawm Ohkawa li al. [37]. Cov tshuaj tiv thaiv sib xyaw suav nrog 0.1 mL ntawm cov ntaub so ntswg homogenate, qhov sib npaug ntawm Sodium Dodecyl Sulfate tov, 0.75 mL ntawm acetic acid, 0.75 mL ntawm thiobarbituric acid, thiab 0.3 mL dej distilled. Cov khoom no tau muab sib xyaw rau hauv ib lub vortex thiab incubated nyob rau hauv ib tug boiling dej da dej rau 1 h ces txias rau chav tsev kub. Kaum 0.5 mL ntawm cov dej distilled thiab 2.5 mL n-butanol tau ntxiv rau txhua lub raj thiab sib xyaw ua ke nrog lub vortex ces tig rau hauv ib lub centrifuge ntawm 2,500 × g rau 10 min. Absorbance tau nyeem ntawm 532 nm.

2.5 Kev txiav txim siab Hormones

2.5.1 Testosterone

Testosterone tau txiav txim siab siv cov khoom siv Testosterone Enzyme-linked immunosorbent assay (ELISA) (Cat. No: ADI-900-065; Enzo Life Sciences, NY, USA), Testosterone yog lub ntsiab androgen uas cuam tshuam rau kev loj hlob ntawm kev sib deev thawj thiab theem nrab. kev sib deev tsav. 100 µL ntawm tus qauv diluent (kev ntsuam xyuas tsis 3 lossis cov ntaub so ntswg kab lis kev cai) tau muab tso rau hauv qhov tsis tshwj xeeb khi thiab qhov siab tshaj plaws (0 pg / mL standard) qhov dej. Tom qab ntawd 100 µL ntawm tus qauv # 1 txog # 5 rau hauv qhov dej tsim nyog. Cov qauv (100 µL) tau muab tso rau hauv qhov dej uas tsim nyog thiab 50 µL ntawm qhov ntsuas tsis tau 3 rau hauv cov qhov dej tsis tshwj xeeb. Tsis tas li ntawd, 50 µL ntawm cov tshuaj tiv thaiv kab mob daj tau ntxiv rau hauv txhua lub qhov dej, tshwj tsis yog qhov khoob, tag nrho cov dej num, thiab cov qhov dej tsis tshwj xeeb. Lub incubation ntawm lub phaj ntawm chav tsev kub ntawm lub phaj shaker rau 1 h ntawm 500 rpm. Tom qab ntawd, 50 µL ntawm xiav conjugate tau ntxiv rau hauv txhua qhov dej, tshwj tsis yog rau tag nrho cov dej num thiab qhov dej tsis muaj. Tom qab tshem lub qhov dej los ntawm cov ntsiab lus, ntxuav tau ua tiav los ntawm kev ntxiv 400 µL ntawm cov tshuaj ntxuav rau txhua qhov dej 3 zaug. Blue conjugate (5 µL) tau ntxiv rau tag nrho cov dej ua haujlwm thiab 200 µL ntawm cov tshuaj p-nitrophenyl phosphate tau ntxiv rau txhua qhov dej thiab incubated ntawm 37 degree rau 1 h yam tsis muaj co. Kev nyeem ntawv ntawm lub phaj ntawm 405 nm tau ua tiav tom qab ntxiv 50 µL ntawm cov tshuaj nres rau txhua qhov dej [12, 48].

2.5.2 Estradiol Cov

Estradiol tau txiav txim siab siv cov khoom siv estradiol ELIZA (Cat. No: 501890; Cayman Chemical, Michigan, USA) raws li cov chaw tsim khoom cov txheej txheem. Lub hauv paus ntsiab lus ntawm cov tshuaj tiv thaiv no yog raws li qhov concentration ntawm estradiol acetylcholinesterase (AChE) tracer yog tas li, tab sis cov haiv neeg estradiol concentration txawv yog li ntawd tus nqi ntawm estradiol acetylcholinesterase (AChE) tracer uas khi rau estradiol antiserum yog inversely proportional rau cov concentration ntawm haiv neeg. hauv qhov dej. 100 µL ELISA tsis tau ntxiv rau cov tsis muaj kev sib khi thiab qhov siab tshaj plaws khi. Ntxiv 50 µL los ntawm raj #8 mus rau ob qho tib si tus qauv qis tshaj plaws (Standard 8) thiab 50 µL los ntawm raj #7 mus rau txhua qhov dej txheem tom ntej (Standard 7). Ib qho piv txwv (50 µL) tau ntxiv rau txhua qhov dej. Estradiol AChE Tracer (50 µL) tau ntxiv rau txhua qhov kev ua ub no thiab qhov dej tsis muaj. Tom qab ntawd Estradiol ELISA antiserum (50 µL) tau ntxiv rau txhua qhov dej tshwj tsis yog rau tag nrho cov haujlwm, tsis muaj kev khi tshwj xeeb, thiab cov qhov dej tsis muaj. Tom qab orbital shaker incubation (2 h ntawm chav tsev kub), qhov nqus tau ntsuas ntawm 414 nm [52].

2.6 Kev txiav txim siab ntawm lub cev muaj peev xwm enzymes

2.6.1 Myeloperoxidase (MPO, EC 1.11.2.2)

Ib chav tsev ntawm MPO kev ua ub no txhais tau tias yog cov enzyme uas hydrolyzes substrate thiab tsim taurine chloramine kom haus 1.0 μmole ntawm TNB ib feeb ntawm 25 degree . MPO tau txiav txim siab siv cov khoom kuaj mob myeloperoxidase colorimetric (Cat. No: MAK068; Sigma-Aldrich, MO, USA). Lub absorbance yog ntsuas ntawm 412 nm.

2.6.2 Adenosine deaminase (ADA, EC 3.5.4.4)

ADA yog ib qho enzyme uas catalyzes hloov pauv ntawm adenosine thiab 2'-deoxyadenosine rau inosine thiab 2'-deoxyinosine. ADA tau txiav txim siab raws li cov hauv paus ntsiab lus ntawm cov ntawv tshaj tawm kev tshaj lij adenosine deaminase cov khoom siv tshuaj ntsuam xyuas (Cat. No: MAK400; Merck KGaA, Darmstadt, Lub teb chaws Yelemees). Ib chav tsev ntawm adenosine deaminase yog tus nqi ntawm cov enzyme uas hydrolyzes adenosine kom tawm los 1.0 μmol ntawm inosine ib feeb ntawm 37 degree. Cov reagents yog 1 × ADA assay buffer (41 μL), ADA Converter (2 μL), ADA tsim tawm (2 μL), thiab ADA substrate (5 μL).

2.7 Qhov sib txawv hemocyte suav

Lub hemolymph tau sau los ntawm cov qwj (tswj, nthuav tawm) raws li Sminia [47]. Tom qab tau kov lub taub hau-ko taw los ntawm Pasteur pipette, tus qwj raug yuam kom rov qab tob rau hauv nws lub plhaub thiab extrude hemolymph, uas tau sau rau qhov sib txawv hemocyte suav. Hemolymph tau smeared rau ntawm lub tshuab tsom iav kom sib kis. Tom qab kho hauv 99.8 feem pua ​​​​methanol rau 5 min, tus swb tau tig ntawm 45 degree lub kaum sab xis kom qhuav ntawm chav tsev kub thiab stained nrog Giemsa stain rau 20 min. Qhov sib txawv ntawm hemocytes tau txiav txim siab ua kom sib npaug rau suav 100.

2.8 Kev tshawb nrhiav histological

Kev kuaj mob histological tau ua tiav tom qab 7 hnub ntawm kev kis mus rau 8 thiab 16 µL / L RIPEX. Cov qog nqaij hlav thiab ovotestis tau muab tshem tawm thiab kho tam sim hauv Bouin᾿s kua. Tom qab 1 hnub ntawm kev txhim kho, cov qauv tau muab lub cev qhuav dej los ntawm cov dej cawv thiab tshem tawm hauv xylene. Paraffin wax blocks tau ua [42] Kaum Pawg. (5-7 μm thickness) raug txiav thiab stained nrog hematoxylin thiab eosin (Mayer's H thiab E). Kev zas xim yog ua raws li kev ntxuav zoo nrog dej kais dej. Cov seem histological tau yees duab siv lub koob yees duab-automated (Optika, Ltalis).

2.9 Kev txheeb cais

Cov ntaub ntawv tau nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv thiab txheeb xyuas los ntawm kev txheeb xyuas software ntawm social sciences (SPSS; IBM Corp. Armonk, NY, USA). Ob txoj kev ANOVA tsom xam ntawm qhov sib txawv tau siv los txheeb xyuas qhov sib txawv ntawm cov tswj thiab cov pab pawg raug, ntawm qhov sib txawv ntawm qhov sib txawv thiab cov ntsiab lus ntawm lub sijhawm raug. Qib ntawm qhov tseem ceeb tau teeb tsa ntawm p Tsawg dua lossis sib npaug rau 0.05.

Lub mechanism ntawm Cistanche boosts testosterone nyhuv

Cistanche tau pom los txhawb qib testosterone hauv ntau txoj hauv kev. Ua ntej, nws muaj cov tebchaw hu ua echinacoside thiab acteoside, uas tau pom tias txhim khu kev tsim cov tshuaj luteinizing hormone (LH) hauv pituitary caj pas. LH txhawb nqa Leydig hlwb hauv cov qe ntshav los tsim cov testosterone. Cistanche kuj tseem muaj cov polysaccharides thiab phenylethanoid glycosides, uas tau pom tias muaj cov tshuaj tua kab mob antioxidant thiab tiv thaiv kab mob. Qhov no tuaj yeem pab txo qis oxidative kev nyuaj siab thiab mob hauv cov qog nqaij hlav, uas tuaj yeem cuam tshuam rau testosterone ntau lawm Tsis tas li ntawd, Cistanche tau pom tias yuav ua rau kom muaj kev qhia ntawm cov noob koom nrog hauv testosterone synthesis thiab txo cov haujlwm ntawm cov enzymes uas ua rau testosterone, xws li {{1} }alpha-reductase. Zuag qhia tag nrho, kev sib xyaw ua ke ntawm cov txheej txheem no tau xav tias yuav pab txhawb rau Cistanche's testosterone-boosting teebmeem.

Hoda H. Abdel-Azeem1*, Azza H. Mohamed1 thiab Mohamed R. Habib2