Inqaku
I-methylene blue nokudluliselwa kwemiqondiso yokudumba: ii-inflammasome, i-IL-6, ne-STAT3
Masithi iilebhu ezintathu zinika ingxelo yokuba kwaumchiza omnye ozuba unciphisa ukudumba. Enye ibonisa i-IL-1beta encinane evela kwii-macrophage. Enye ibonisa i-enzyme ye-iNOS encinane kwakolo hlobo lweeseli. Enye ibonisa i-IL-6 encinane egazini nokusebenza okuncinane kwe-STAT3 engqondweni. Kuyahenda ukudibanisa ezi ziphumo zintathu zibe libango elinye. Oko kudibanisa kufihla oko kufunyenweyo.
Isiphumo ngasinye sinxulumene nendlela eyahlukileyo yokudluliselwa kwemiqondiso, enesivuseleli sayo, inyathelo lokudityaniswa, nomlinganiselo wesiphumo. I-methylene blue ichaphazela indlela nganye kwindawo eyahlukileyo. Ukuba nayiphi na kwezi iphucula isiphumo sempilo ngumbuzo owahlukileyo, oxutyushwa kwisishwankathelo sethu se-methylene blue nokudumba. Eli phepha ligxile kwiindlela zokudluliselwa kwemiqondiso.
Indlela 1: ukudityaniswa kwe-inflammasome ne-caspase-1
I-inflammasome sisakhiwo esixhasayo seeproteni esakhiwa xa sifuneka ngaphakathi kwiseli ye-myeloid. Amalungu ayo sisiva, iproteni edibanisayo ebizwa ngokuba yi-ASC, ne-enzyme ephumeza impendulo ebizwa ngokuba yi-caspase-1. Xa sele edityanisiwe, i-caspase-1 isika i-pro-IL-1beta ne-pro-IL-18 zibe ziimo zazo ezikhutshelwa ngaphandle kweseli, yaye inokubangela i-pyroptosis, uhlobo lokufa kweseli oluhamba nokudumba. Isiva sichaza uhlobo: i-NLRP3 isabela kwiikristale nakwiityhefu ezenza imingxuma, ezifana ne-nigericin ne-ATP; i-NLRC4 isabela kwi-flagellin yeebhaktheriya; i-AIM2 isabela kwi-DNA enemisonto emibini ekwi-cytosol; yaye indlela engeyiyo eyesiqhelo isabela kwi-lipopolysaccharide engaphakathi kwiseli.
Ukuvuselelwa kufuna imiqondiso emibini. Umqondiso wokuqala ngowokulungiselela: i-ligand ye-receptor efana ne-Toll iqhuba ukukhutshelwa kolwazi lwemfuza lwe-NLRP3 ne-pro-IL-1beta oluxhomekeke kwi-NF-kB, ukuze amalungu abe khona. Umqondiso wesibini ngowokudityaniswa: isivuseleli sesibini sibangela ukuba iimolekyuli ze-ASC zidibane zibe ngamachaphaza, i-caspase-1 isikwe ibe sisiqwenga sayo se-p20, kuze kukhutshwe i-IL-1beta p17 evuthiweyo. Iintlobo zeoksijini ezisebenzayo ezivela kwi-mitochondria nokungeniswa kweekristale kwiseli zizinto ezaziwayo ezinegalelo kumqondiso wesibini we-NLRP3.
Uphononongo luka-2017 lwee-macrophage malunga nokuthintelwa kweentlobo ezininzi zee-inflammasome lwavavanya i-methylene blue kuyo yomibini le miqondiso. Kwii-macrophage zomongo wethambo lempuku ezazilungiselelwe nge-lipopolysaccharide, i-nigericin, i-ATP, neekristale ze-monosodium urate nganye zabangela ukukhutshwa kwe-IL-1beta ne-caspase-1 kwakunye nokwakheka kwamachaphaza e-ASC. I-methylene blue kwi-20 ukuya kwi-200 micromolar yazinciphisa zontathu ngendlela exhomekeke kwidosi, ngaphandle kokubulala iiseli. I-flagellin ne-Salmonella ze-NLRC4, i-DNA enemisonto emibini ne-Listeria ze-AIM2, kwakunye ne-lipopolysaccharide efakwe ngaphakathi kweeseli okanye i-E. coli ephilayo yendlela engeyiyo eyesiqhelo, zabonisa ipateni efanayo. Umchiza uwodwa awuzange ubangele ukukhutshwa, yaye iipleyiti zolawulo azibonisanga mpembelelo yokuthintela iibhaktheriya. Ngoko isiphumo sinxulumene nempendulo yomzimba womamkeli.
Kumanqanaba angaphambili ale ndlela, kwalinganiswa izinto ezintathu ezichaphazelekayo. I-superoxide ye-mitochondria ebangelwe yi-rotenone yancipha nge-methylene blue, kwaza ukukhutshwa kwe-IL-1beta okubangelwe yi-rotenone nako kwancipha. Ukungeniswa kwamasuntswana abengezelayo kwiseli kwancipha, nto leyo ehambelana nokuncipha kwe-phagocytosis ngaphambi kokuba iikristale zibonwe sisiva. Inkqubo yokunika ingxelo eqhutywa ngummandla okhuthaza ukukhutshelwa kolwazi lwemfuza lwe-NLRP3, oneendawo ezimbini ze-NF-kB, yabonisa ukusebenza okuphantsi nge-methylene blue. Kodwa ummandla okhuthazayo ofinyeziweyo ongenazo ezo ndawo awuzange usabele. Kwityhubhu engenazo iiseli, i-methylene blue yanciphisa ngokuthe ngqo ukusebenza kwe-caspase-1 yomntu eveliswe ngobuchule bokudibanisa imfuza. Ukongeza umchiza kuphela ngexesha lokulungiselela okanye kuphela ngexesha lokuvuselela kwakusanciphisa ukukhutshwa kwe-IL-1beta, i-IL-18, ne-caspase-1, ngoko omabini la manyathelo ayabandakanyeka.
Uvavanyo lweempuku lwahambelana nolu khetho lweziphumo. Idosi ebulalayo ye-lipopolysaccharide yabulala iimpuku zolawulo kwiiyure ezimbalwa, ngoxa i-methylene blue yaphucula ukusinda; i-IL-1beta ekumngxuma wesisu yancipha, kodwa i-IL-6 kolu lwelo lunye ayizange inciphe. Kwimodeli yokudumba kwenwebu yomngxuma wesisu okubangelwe yi-Listeria, i-IL-1beta ekumngxuma wesisu yancipha ngoxa amanani ewonke eeseli ayengatshintshanga. Ii-monocyte zomntu ze-THP-1 zaziziphatha njengee-macrophage zempuku kuzo zonke iintlobo ezine zee-inflammasome. Isiphumo se-inflammasome sichaza ukuvuthwa kwe-IL-1beta ne-IL-18, hayi zonke ii-cytokine. Kungenxa yoko le nto ukumila kweeseli zokhuselo lomzimba ezisengqondweni kuvavanyo lwe-microglia nee-astrocyte kuphendula umbuzo owahlukileyo kulowo wokudityaniswa kwesakhiwo esixhasayo.
Indlela 2: ukukhutshelwa kolwazi lwemfuza lwe-iNOS ngokubopha kwe-NF-kB ne-STAT1
I-nitric oxide synthase evuselelwayo, okanye i-iNOS, iphantse ingabikho xa iseli iphumle, yaye ulwazi lwayo lwemfuza lukhutshelwa xa lufuneka. I-lipopolysaccharide iqhuba oku ikakhulu ngokuchithwa kwe-IkB-alpha, kulandelwe kukongezwa kweqela le-phosphate kwi-NF-kB nokungena kwayo kwinucleus. I-interferon-gamma iqhuba oku ikakhulu ngokongezwa kweqela le-phosphate kwi-STAT1 yi-Janus kinase, kulandelwe kukungena kwe-STAT1 kwinucleus. Iproteni nganye ke ibopha kwindawo yayo kummandla okhuthaza ukukhutshelwa kolwazi lwemfuza lwe-iNOS. Ukusebenza kwe-enzyme nokuvuselelwa kofuzo ziindawo ezimbini ezahlukeneyo zolawulo, yaye ichiza linokusebenza kwenye okanye kuzo zombini.
Uphononongo luka-2015 lokuvuselelwa kwe-iNOS kwii-macrophage nakwiimpuku ezine-endotoxin egazini lwafumanisa ukuba impembelelo ikwinqanaba lomyalezo wemfuza. Kwiiseli ze-RAW 264.7 nakwii-macrophage ezithathwe ngqo kwiimpuku, i-1 micromolar ye-methylene blue enikwe imizuzu eyi-30 ngaphambi kwe-lipopolysaccharide, i-interferon-gamma, okanye zombini, yanciphisa iproteni ye-iNOS ne-mRNA kwiiyure eziyi-8 ukuya kweziyi-24. Kwakukho ne-nitrite encinane kulwelo lokukhulisa iiseli, ngaphandle kokuncipha kokuphila kweeseli. Kwiimpuku ezinikwe i-25 mg nge-kg ye-lipopolysaccharide, idosi enye ye-5 mg nge-kg enikwe iyure enye ngaphambili yanciphisa iproteni ye-iNOS ne-mRNA emiphungeni, esibindini, nasentliziyweni.
Inkcukacha eyahlulayo koko kungazange kutshintshe. Ukuchithwa kwe-IkB-alpha, ukongezwa kweqela le-phosphate kwi-NF-kB nakwi-STAT1, nokuqokelelana kwayo nayiphi na kwezi proteni kwinucleus kwahlala kungachaphazelekanga. Uphazamiseko lwenzeka kwinyathelo elilandelayo: izinto ezikhutshwe kwinucleus zabonisa ukubopha okuncinane kwe-NF-kB kwindawo yayo ye-DNA emva kwe-lipopolysaccharide, nokubopha okuncinane kwe-STAT1 emva kwe-interferon-gamma. Uvavanyo lokuhlukanisa i-chromatin kusetyenziswa izilwa-buhlungu lwaqinisekisa ubukho obuncinane bazo zombini ezi proteni kummandla okhuthaza ukukhutshelwa kolwazi lwemfuza lwe-iNOS ngaphakathi kweeseli. I-tumor necrosis factor alpha, enommandla okhuthazayo osebenzisa i-NF-kB nawo, nayo yancipha. Ezi proteni zavuselelwa zaza zafika, kodwa ukubopha kwazo kwi-DNA ekujoliswe kuyo kwakubuthathaka.
Qaphela umahluko kule ndlela nakwindlela 1. Oku kuchaphazela ukubopha kweproteni kwi-DNA, kusebenzisa i-STAT1 kungekhona i-STAT3, yaye kulinganisa i-mRNA, iproteni, ne-nitrite kunamachaphaza e-ASC namaqhekeza e-caspase-1. Imephu yethu yeendawo ekujoliswa kuzo yi-methylene blue kwiimodeli zeeseli igcina eli nqanaba lokukhutshelwa kolwazi lwemfuza lahlukile kwimilinganiselo ethe ngqo yee-enzyme ne-mitochondria.
Indlela 3: i-IL-6 kwiserum nokuvuselelwa kwe-STAT3 kwizicubu
I-IL-6 idlulisa imiqondiso ngosapho lwee-receptor ze-gp130 iye kuthotho lwamanyathelo ukusuka kwi-Janus kinase ukuya kwi-STAT3. I-STAT3 eyongezwe iqela le-phosphate idibana ngababini, ingene kwinucleus, ize iqhube iinkqubo zokudumba. Kwangaxeshanye, i-IL-6 ngokwayo iyinto ekujoliswa kuyo yi-NF-kB, ngoko ezi zimbini zenza umjikelo wengxelo obizwa rhoqo ngokuba ngumgca we-IL-6 ukuya kwi-STAT3. Imilinganiselo eqhelekileyo yi-IL-6 kwiserum kwinqanaba elingaphambili, umlinganiselo we-STAT3 eyongezwe iqela le-phosphate xa ithelekiswa ne-STAT3 iyonke, nee-enzyme eziphumeza impendulo ezifana ne-iNOS ne-COX2 kumanqanaba alandelayo.
Uphononongo luka-2023 lweempuku nge-IL-6 ne-STAT3 emva kwe-lipopolysaccharide lwalinganisa lo mgca kwiindawo ezahlukeneyo zomzimba. Iimpuku eziyinkunzi ze-C57BL/6 zanikwa i-1 mg nge-kg ye-lipopolysaccharide yonke imihla kangangeentsuku ezintathu, kunye ne-5 okanye i-10 mg nge-kg ye-methylene blue kwimizuzu eyi-30 emva kwedosi nganye. Ngosuku lwesithathu, i-IL-6 kwiserum yayisezantsi kuwo omabini amaqela e-methylene blue kunakwiqela elanikwa i-lipopolysaccharide kuphela, ngoxa uninzi lwezinye ii-cytokine ezazilinganiswa lungazange lutshintshe ngonyango. Imilinganiselo ye-STAT3 eyongezwe iqela le-phosphate xa ithelekiswa ne-STAT3 iyonke yayisezantsi kwi-cortex, kwi-hippocampus, nakwisikhumba sendlebe. Kwakhona kwakukho ukuphawulwa okuncinane kwe-microglia nge-Iba-1, i-iNOS ne-COX2 ezisezantsi, nokuncipha ngokuyinxenye kokulahleka kobunzima.
Kukho izilumkiso ezimbini ezifanelekileyo apha. I-IL-6 esezantsi kwiserum kunye nokongezwa okuncinane kwamaqela e-phosphate kwi-STAT3 yezicubu kuyahambelana nokulungiswa kwalo mgca, kodwa akubonisi ngokukodwa eyona ndawo iphambili echaphazelekayo. Isizathu kukuba i-cytokine encinane kwinqanaba elingaphambili, imiqondiso embalwa evela kwi-receptor, okanye ukutshintsha kokusebenza kwe-phosphatase nganye inokuvelisa kwaloo mlinganiselo. Kwaye ubunzima obulinganiswa kwiintsuku ezintathu zovavanyo oluqatha ngumlinganiselo wesilwanyana siphela, hayi inqaku leempawu okanye lokuthomalala kwesifo. Oku kongeza umxholo ongaphaya kovavanyo lwe-blot, ngaphandle kokuguqula isiphumo semiqondiso sibe libango lonyango. Imichiza eyahlukileyo elawula iinkqubo ze-redox kwi-mitochondria isebenza ngokunxuseneyo nale nkcazelo yee-cytokine, yaye akufanele idityaniswe nayo.
Ukuthelekiswa kweendlela: iindawo ezichaphazelekayo nemilinganiselo yeziphumo
| Indlela | Isivuseleli kula maphepha | Apho i-methylene blue isebenza khona | Umlinganiselo wesiphumo otshintshileyo |
|---|---|---|---|
| Ukudityaniswa kwe-inflammasome | I-nigericin, i-ATP, iikristale ze-urate, i-flagellin, i-DNA, i-lipopolysaccharide ekwi-cytosol | Ukukhutshelwa okuncinane kolwazi lwemfuza ngexesha lokulungiselela, i-ROS encinane ye-mitochondria, i-phagocytosis encinane, ukudityaniswa okuncinane kwe-ASC, ukuthintelwa ngokuthe ngqo kwe-caspase-1 | I-caspase-1 p20 encinane, i-IL-1beta ne-IL-18 ezivuthiweyo ezimbalwa, amachaphaza e-ASC ambalwa |
| Ukukhutshelwa kolwazi lwemfuza lwe-iNOS | I-lipopolysaccharide nge-NF-kB; i-interferon-gamma nge-STAT1 | Ukubopha okuncinane kwe-NF-kB ne-STAT1 kwi-DNA yommandla okhuthazayo nangona ukuvuselelwa nokungena kwinucleus kuqhelekile | I-mRNA neproteni ye-iNOS ezimbalwa, i-nitrite encinane |
| Umgca we-IL-6 ukuya kwi-STAT3 | I-lipopolysaccharide kangangeentsuku ezintathu kwiimpuku | I-IL-6 esezantsi kwiserum kunye nokongezwa okuncinane kwamaqela e-phosphate kwi-STAT3 yezicubu | Umlinganiselo osezantsi we-pSTAT3 xa ithelekiswa ne-STAT3 kwi-cortex, kwi-hippocampus, nakwisikhumba; i-iNOS, i-COX2, ne-Iba-1 ezisezantsi |
Le theyibhile ithintela ukudluliselwa kwezigqibo ezintathu ezingachananga. Isiphumo samachaphaza e-ASC asingqineli isiphumo se-STAT3. Isiphumo sokubopha kwe-STAT1 kummandla okhuthaza ukukhutshelwa kolwazi lwemfuza lwe-iNOS asingqineli isiphumo se-STAT3, nangona zombini ezi proteni zingamalungu osapho lwe-STAT. Isiphumo se-IL-6 kwiserum asingqineli ukuncipha kokudityaniswa kwe-inflammasome: kuvavanyo lomngxuma wesisu i-IL-1beta yancipha ngoxa i-IL-6 ingazange inciphe. Umqolo ngamnye ufuna isivuseleli sawo nomlinganiselo wawo wesiphumo. Kungenxa yoko le nto ukufunda uphononongo lwe-methylene blue lweeseli, lwezilwanyana, nolwezonyango kufuna ukuba ibango ngalinye lingqinelaniswe nenqanaba elilinganisiweyo.
Akukho nanye kwezi emisela idosi, ukhuseleko, okanye inzuzo ebantwini. Iimodeli ze-lipopolysaccharide zizivuseleli ezilawulwayo zokuvavanya indlela yokusebenza, hayi iikopi zezifo ezingapheliyo. Kwaye ukunika idosi kunye nesivuseleli kuvavanya ukuthintelwa kotshintsho olubangelweyo, kunokubuyiselwa umva kwesifo esele sikhona. Gcina le nkcazelo yeendlela zokusebenza yahlukile kwiziphumo kwizigulana, uze ugwebe naliphi na ibango lempilo ngokophononongo lwezigulana oluxhasa elo bango.