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Kwayoyin Ban-Lan-Gen suna rage yawan sake kamuwa da cutar dextran sodium sulfate a cikin beraye ta hanyar daidaita ƙwayoyin cuta na hanji da kuma dawo da samar da SCFA Derived-GLP-1 na hanji.
Jiao Peng,1-3,*Li Xi,4,*Zheng Lin,3,5 Duan Lifang,1 Gao Zhengxian,2,5 Diehu,1 Li Jie,6 Li Xiaofeng,6 Shen Xiangchun,5 Xiao Haitao21 Jami'ar Peking Asibitin Shenzhen Sashen Magunguna, Shenzhen, Jamhuriyar Jama'ar China; 2 Cibiyar Kimiyyar Lafiya ta Jami'ar Shenzhen Makarantar Magunguna, Shenzhen, Jamhuriyar Jama'ar China; 3 Cibiyar Bincike ta Fasaha ta Injiniya ta Jami'ar Likitanci ta Guizhou Cibiyar Nazarin Magungunan Kabilanci da Ci gaban Magungunan Gargajiya da Amfani da Magungunan Sin Ma'aikatar Ilimi, Dakin Gwaji na Lardin Guizhou na Magunguna, Jami'ar Likitanci ta Guizhou, Guiyang, Jamhuriyar Jama'ar China; 4 Sashen Gastroenterology, Asibitin Jami'ar Peking Shenzhen, Shenzhen, Jamhuriyar Jama'ar China; 5 Makarantar Magunguna, Jami'ar Likitanci ta Guizhou, Dakin Gwaji na Jiha na Ayyukan Shuke-shuke da Aiwatarwa, Guiyang; 6 Sashen Magungunan Dakunan Gwaje-gwaje, Asibitin Shenzhen na Jami'ar Peking, Shenzhen, China [email protected] Shen Xiangchun, Makarantar Magunguna, Jami'ar Likitancin Guizhou, Guizhou, Jamhuriyar Jama'ar China, 550004, Imel [email protected] Manufa: Maganin da aka yi da GLP-1 sabon zaɓi ne na magani don cututtukan hanji. Kwayoyin Ban-Lan-Gen (BLG) sanannen tsari ne na rigakafi na TCM wanda ke nuna yuwuwar aikin hana kumburi a cikin maganin yanayi daban-daban na kumburi. Duk da haka, tasirinsa na hana kumburi akan colitis da tsarin aikinsa har yanzu ba a fayyace shi ba. HANYOYI: Don kafa colitis mai sake dawowa da dextran sodium sulfate (DSS) a cikin beraye. An yi alamun ayyukan cututtuka, alamun rauni na tarihi, da matakan cytokine masu kumburi don tantance tasirin kariya na BLG. Tasirin BLG akan microbiota na hanji da hanji an siffanta su da matakan GLP-1 na jini da bayyanar Gcg na hanji, GPR41, da GRP43, abun da ke cikin microbiota na hanji, matakan SCFAs na najasa, da Fitowar GLP-1 daga ƙwayoyin epithelial na hanji na farko na SCFA wanda aka samo daga SCFA daga GLP-1. Sakamako: Maganin BLG ya rage asarar nauyin jiki sosai, DAI, rage girman hanji, lalacewar nama na hanji, da matakan cytokine masu kumburi na TNF-α, IL-1β, da IL-6 a cikin kyallen hanji. Bugu da ƙari, maganin BLG zai iya dawo da bayyanar Gcg na hanji, GPR41 da GRP43 da matakan GLP-1 a cikin ƙwayoyin colitis, da kuma ta hanyar ƙara yawan ƙwayoyin cuta masu samar da SCFA kamar Akkermansia da Prevotellaceae_UCG-001, da rage yawan ƙwayoyin cuta kamar Eubacterium_xylanophilum_group, Ruminokoccaceae_UCG-014, Intestinimonas da Oscillibacter. Bugu da ƙari, maganin BLG na iya ƙara yawan SCFAs a cikin najasar beraye na colitis. A lokaci guda, gwaje-gwajen in vitro sun kuma nuna cewa cirewar najasar beraye da aka yi wa magani da BLG na iya ƙarfafa ƙananan ƙwayoyin epithelial na hanji na farko na Murine suna fitar da GLP-1. Kammalawa: Waɗannan binciken sun nuna cewa BLG yana da tasirin hana kamuwa da cutar colitis. BLG yana da yuwuwar haɓaka shi azaman magani, aƙalla wani ɓangare ta hanyar daidaita ƙwayoyin cuta na hanji da dawo da samar da GLP-1 daga SCFA daga hanji Magunguna masu kyau don ciwon colitis mai sake dawowa akai-akai. Kalmomi masu mahimmanci: colitis, Ban-Lan-Gen granules, gut microbiota, short-chain fatty acids, GLP-1
Ulcerative colitis (UC) cuta ce mai kumburi ta hanji da dubura wadda ke da alaƙa da gudawa mai yawan faruwa, ciwon ciki, raguwar nauyi, da kuma zubar jini a cikin hanji.1 Kwanan nan, yawan UC yana ƙaruwa a ƙasashen da a da ba su da yawan kamuwa da cutar, ciki har da China, tare da karuwar shaharar salon rayuwa na ƙasashen Yamma.2 Wannan ƙaruwar tana haifar da manyan matsaloli ga lafiyar jama'a kuma tana da mummunan tasiri ga ikon marasa lafiya na aiki da ingancin rayuwa. Abin lura shi ne, cutar UC har yanzu ba a fayyace ta ba, amma gabaɗaya an yarda cewa kwayoyin halitta, abubuwan muhalli, ƙwayoyin cuta na hanji, da tsarin garkuwar jiki duk suna ba da gudummawa ga ci gaban UC.3 Har yanzu, babu magani ga UC, kuma manufar magani ita ce a kula da alamun asibiti, haifar da da kuma kula da gafara, haɓaka warkar da mucosal, da rage sake dawowa. Magungunan gargajiya sun haɗa da aminosalicylates, corticosteroids, immunosuppressants, da biologics. Duk da haka, waɗannan magunguna ba za su iya cimma tasirin da ake so ba saboda illolinsu daban-daban.4 Kwanan nan, bincike da yawa sun nuna cewa maganin gargajiya na kasar Sin (TCM) ya nuna babban yuwuwar taimakawa wajen rage UC tare da ƙarancin guba, yana nuna cewa Ci gaban sabbin hanyoyin magance cutar TCM dabarar magani ce mai kyau ga UC.5-7​ ...
Banlangen Granules (BLG) wani shiri ne na gargajiya na kasar Sin wanda aka yi daga ruwan da aka samo daga tushen Banlangen.8 Baya ga ingancinsa na rigakafi, BLG yana nuna yuwuwar hana kumburi wajen magance cututtuka daban-daban na kumburi.9,10 Bugu da ƙari, an ware glucosinolates (R,S-goitrin, progoitrin, epiprorubin da glucoside kuma an gano su daga ruwan da aka samo daga Radix isatidis) da nucleosides (hypoxanthine, adenosine, uridine da guanosine) da kuma indigo alkaloids kamar indigo da indirubin.11,12 Nazarin da aka yi a baya sun tabbatar da cewa sinadaran adenosine, uridine da indirubin suna nuna tasirin hana kumburi a cikin nau'ikan cututtukan colitis daban-daban na dabbobi.13-17 Duk da haka, babu wani bincike da aka gudanar bisa shaida don tantance ingancin BLG a cikin ciwon colitis. A cikin wannan binciken, mun binciki tasirin kariya na BLG akan ciwon colitis na dextran sodium sulfate (DSS) wanda ke haifar da sake dawowar ciwon colitis a cikin beraye C57BL/6 kuma an gano cewa shan BLG ta baki ya rage yawan sake dawowar hanji da DSS ke haifarwa a cikin beraye. Kumburi, hanyoyin da ake amfani da su wajen tsara abinci suna da alaƙa da daidaita ƙwayoyin cuta na hanji da kuma dawo da samar da glucagon-like peptide-1 (GLP-1) da aka samo daga hanji.
An sayi ƙwayoyin BLG (ba su da sukari, Z11020357 da NMPA ta amince da shi; Beijing Tongrentang Technology Development Co., Ltd., Beijing, China; lambar rukuni: 20110966) daga shagunan magani. An sayi DSS (Nauyin ƙwayoyin halitta: 36,000–50,000 Daltons) daga MP Biologicals (Santa Ana, Amurka). An sayi Sulfasalazine (SASP) (≥ 98% tsarki), hematoxylin da eosin daga Sigma-Aldrich (St. Louis, MO, Amurka). An sayi kayan gwajin Elisa na linzamin kwamfuta TNF-α, IL-1β da IL-6 luminex daga tsarin R&D (Minneapolis, MN, Amurka). An sayi acetic acid, propionic acid, da butyric acid daga Aladdin Industries (Shanghai, China). An sayi 2-Ethylbutyric acid daga Merck KGaA (Darmstadt, Jamus).
An sayi beraye maza 'yan makonni 6-8 masu nauyin C57BL/6 (nauyin jiki 18-22 g) daga Kamfanin Beijing Wetahe Laboratory Animal Technology Co., Ltd. (Beijing, China) kuma an ajiye su a cikin yanayi mai zafi na 22 ± 2 °C tare da haske/zagaye mai duhu na awanni 12. An ciyar da beraye abinci na yau da kullun tare da samun ruwan sha kyauta na tsawon mako guda don daidaitawa da sabon muhalli. Daga nan aka raba beraye zuwa ƙungiyoyi huɗu bazuwar: ƙungiyar kulawa, ƙungiyar samfurin DSS, ƙungiyar da aka yi wa magani da SASP (200 mg/kg, na baki) da ƙungiyar da aka yi wa magani da BLG (1 g/kg, na baki). Kamar yadda aka nuna a Hoto na 1A, bisa ga bincikenmu na baya, an haifar da cutar colitis ta gwaji mai tsanani ta hanyar zagayowar 1.8% na DSS na tsawon kwanaki 5, sannan aka tace ruwan na tsawon kwanaki 7, bisa ga bincikenmu na baya.18 An yi wa beraye a cikin ƙungiyoyin da aka yi wa magani da SASP da BLG magani da SASP da BLG, bi da bi, kowace rana tun daga ranar 0. Bisa ga gwaje-gwajen farko, adadin An saita BLG a 1 g/kg. A halin yanzu, an saita adadin SASP a 200 mg/Kg bisa ga wallafe-wallafen.4 Ƙungiyoyin sarrafawa da samfurin DSS sun sami ruwa iri ɗaya a duk lokacin gwajin.
Hoto na 1 BLG yana rage ciwon hanji mai sake dawowa wanda DSS ke haifarwa a cikin beraye.(A) Tsarin gwaji na ciwon hanji mai sake dawowa da magani, (B) canjin nauyin jiki, (C) ma'aunin aikin cuta (DAI), (D) tsawon hanji, (E) hoton da ke wakiltar ciwon hanji, (F) Tarin H&E Ciwon hanji (girma, ×100) da (G) ma'aunin histological. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 6).##p < 0.01 ko ###p < 0.001 vs ƙungiyar kulawa (Con); *p < 0.05 ko **p < 0.01 ko ***p < 0.001 vs ƙungiyar DSS.
Ana yin rikodin nauyin jiki, daidaiton bayan gida, da kuma zubar jinin dubura kowace rana. An tantance ma'aunin ayyukan cututtuka (DAI) ta hanyar haɗa yawan nauyin jiki, daidaiton bayan gida, da zubar jinin dubura kamar yadda aka bayyana a baya.19 A ƙarshen gwajin, an kashe dukkan beraye kuma an tattara jini, najasa da hanji don ƙarin gwaje-gwaje.
An gyara kyallen hanjin kuma an saka shi a cikin paraffin. An yi sassan micron 5 kuma an yi masa fenti da hematoxylin-eosin (H&E), sannan aka makantar da shi kuma aka yi masa alama kamar yadda aka bayyana a baya.19
An cire jimillar RNA na kyallen hanji ta hanyar amfani da sinadarin Trizol (Invitrogen, Carlsbad, CA), sannan aka cire cDNA tare da reverse transcriptase (TaKaRa, Kusatsu, Shiga, Japan). An yi amfani da tsarin PCR na ainihin lokaci tare da SYBR Green Master (Roche, Basel, Switzerland). An daidaita kwafin kwayoyin halitta zuwa β-actin kuma an yi nazarin bayanai ta amfani da hanyar 2-ΔΔCT. An nuna jerin firam ɗin kwayoyin halitta a cikin Jadawali na 1.
An yi aikin keɓewa da kuma haɓaka ƙwayoyin epithelial na hanji na farko kamar yadda aka bayyana a baya.20 A takaice, an fara cire ƙwayoyin beraye masu makonni 6-8 bayan an yi musu hadaya ta hanyar karkacewar mahaifa, sannan aka buɗe su a tsayi, aka yi musu magani da Hanks Balanced Salt Solution (HBSS, ba tare da calcium da magnesium ba) sannan aka yanka su zuwa ƙananan guda 0.5-1 mm. Daga baya, an narke kyallen takarda da 0.4 mg/mL collagenase XI (Sigma, Poole, UK) a cikin matsakaicin DMEM kyauta kuma aka centrifuge a 300 xg na tsawon minti 5 a zafin ɗaki. Sake mayar da pellet ɗin a cikin matsakaicin DMEM (an ƙara shi da 10% serum na shanu na tayi, 100 Units/mL penicillin, da 100 µg/mL streptomycin) a 37 °C sannan a ratsa ta raga ta nailan (girman rami ~250 µm). An sanya adadin ƙwayoyin epithelial na hanji a cikin kwano na ƙasan gilashi kuma an saka su da acetic acid, propionic acid, butyric acid, da ruwan da aka cire daga najasa na tsawon awanni 2 a zafin jiki na 37°C, 5% CO2.
An haɗa ƙwayoyin hanji da PBS, kuma an gano matakan cytokines IL-6, TNF-α da IL-1β a cikin kyallen hanji ta amfani da kayan gwajin luminex ELISA (tsarin R&D, Minneapolis, MN, Amurka). Haka kuma, an tantance matakan GLP-1 a cikin jini da kuma hanyar al'ada na ƙwayoyin epithelial na murine colonic na farko ta amfani da kayan aikin ELISA (Bioswamp, Wuhan, China) bisa ga umarnin masana'anta.
An cire jimillar DNA daga najasa ta amfani da kayan cire DNA (Tiangen, China). An auna inganci da adadin DNA a rabon 260 nm/280 nm da 260 nm/230 nm, bi da bi. Bayan haka, ta amfani da kowace DNA da aka cire a matsayin samfuri, an yi amfani da takamaiman firam 338F (ACTCCTACGGGAGGCAGCAG) da 806R (GGACTACHVGGGTWTCTAAT) don faɗaɗa yankunan V3-V4 na kwayar halittar 16S rRNA a yankuna daban-daban. An tsarkake samfuran PCR ta amfani da Kayan Cire Gel na QIAquick (QIAGEN, Jamus), an ƙididdige ta ta hanyar PCR na ainihin lokaci, kuma an jera su ta amfani da dandamalin jerin IlluminaMiseq PE300 (Illumina Inc., CA, Amurka). Don nazarin bioinformatics, an gudanar da sarrafa bayanai bisa ga ka'idojin da aka bayar a baya.21,22 A takaice, yi amfani da Cutadapt (V1.9.1) don tace fayilolin bayyanannu. An tattara OTUs ta amfani da UPARSE (sigar 7.0.1001) tare da yanke kamanceceniya na kashi 97%, kuma an yi amfani da UCHIME don cire jerin chimeric. An yi nazarin abubuwan da ke cikin al'umma da rarraba su ta amfani da mai rarraba RDP (http://rdp.cme.msu.edu/) bisa ga bayanan kwayoyin halittar SILVA ribosomal RNA.
An auna matakan kitse mai gajere (acetic acid, propionic acid, da butyric acid) kamar yadda Tao et al. suka bayyana a baya, tare da wasu gyare-gyare.23 A takaice, an fara dakatar da najasa 100 MG a cikin 0.4 mL na ruwan da aka cire ion, sannan aka biyo baya da 0.1 mL na 50% sulfuric acid da 0.5 mL na 2-ethylbutyric acid (na ciki), sannan aka daidaita shi aka dumama shi a zafin 4°C. Centrifuge a 12,000 rpm na tsawon mintuna 15 a C. An cire ruwan da ke cikinsa da 0.5 mL na ether sannan aka yi masa allura a cikin GC don yin nazari. Don nazarin gas chromatography (GC), an yi nazarin samfurori ta amfani da GC-2010 Plus gas chromatograph (Shimadzu, Inc.) wanda aka sanye da na'urar gano ionization mai harshen wuta (FID). An cimma rabuwa ta amfani da ginshiƙin ZKAT-624, 30 m × 0.53 mm × 0.3 μm (Lanzhou Zhongke Antai Analytical Technology Co., Ltd., China). An samo bayanai ta amfani da software na GC solution (Shimadzu, Inc.). Rabon raba shine 10:1, iskar mai ɗaukar kaya shine nitrogen, kuma yawan kwarara shine 6 mL/min. Girman allura shine 1 μL. Zafin allura da na'urar ganowa shine 300°C. An riƙe zafin tanda a 140°C na mintuna 13.5, sannan aka ƙara zuwa 250°C a ƙimar na 120°C/min; an riƙe zafin jiki na tsawon minti 5.
An gabatar da bayanai a matsayin matsakaicin ± kuskuren daidaito na matsakaicin (SEM). An kimanta mahimmancin bayanai ta hanyar ANOVA ta hanya ɗaya sannan aka biyo bayan gwajin kewayon Duncan mai yawa. An yi amfani da software na GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, Amurka) don duk lissafin kuma an ɗauki p < 0.05 a matsayin mahimmanci a kididdiga.
Sanannen abu ne cewa UC cuta ce ta ciwon hanji mai sake dawowa da ciwon ciki mai tsanani, gudawa da zubar jini. Saboda haka, an kafa cutar colitis mai sake dawowa da ciwon hanji mai tsanani da DSS ke haifarwa a cikin beraye don tantance tasirin maganin BLG (Hoto na 1A). Idan aka kwatanta da ƙungiyar kulawa, beraye a cikin ƙungiyar samfurin DSS sun rage nauyin jiki sosai da kuma DAI mafi girma, kuma waɗannan canje-canjen sun koma baya sosai bayan kwanaki 24 na maganin BLG (Hoto na 1B da C). Rage girman hanji muhimmin alama ce ta UC. Kamar yadda aka nuna a cikin Hotuna na 1D da E, tsawon hanjin beraye da suka karɓi DSS ya ragu sosai, amma an rage su ta hanyar maganin BLG. Daga baya, an gudanar da nazarin histopathological don tantance kumburin hanji. Hotunan da aka yiwa fenti na H&E da sakamakon cututtukan da suka nuna cewa gudanar da DSS ya kawo cikas ga tsarin hanji sosai kuma ya haifar da lalata crypt, yayin da maganin BLG ya rage lalata crypt da maki na cututtuka sosai (Hoto na 1F da G). Musamman ma, tasirin kariya na BLG a kashi na 1 g/Kg ya yi daidai da na SASP a kashi na 200. mg/Kg. Ta hanyar haɗaka, waɗannan binciken sun nuna cewa BLG yana da tasiri wajen rage tsananin ciwon colitis mai tsanani wanda DSS ke haifarwa a cikin beraye.
TNF-α, IL-1β da IL-6 muhimman alamomin kumburi ne na kumburin hanji. Kamar yadda aka nuna a Hoto na 2A, DSS ta haifar da ƙaruwa mai yawa a cikin bayyanar kwayar halittar TNF-α, IL-1β da IL-6 a cikin hanji idan aka kwatanta da ƙungiyar kulawa. Gudanar da BLG na iya mayar da waɗannan canje-canjen da DSS ta haifar sosai. Na gaba, mun yi amfani da ELISA don tantance matakan cytokines masu kumburi TNF-α, IL-1β, da IL-6 a cikin kyallen hanji. Sakamakon ya kuma nuna cewa matakan hanji na TNF-α, IL-1β, da IL-6 sun ƙaru sosai a cikin beraye da aka yi wa magani da DSS, yayin da maganin BLG ya rage waɗannan ƙaruwar (Hoto na 2B).
Hoto na 2 BLG yana hana bayyanar kwayoyin halitta da samar da cytokines masu hana kumburi TNF-α, IL-1β da IL-6 a cikin hanjin beraye da aka yi wa magani da DSS.(A) Bayyanar kwayar halittar hanji ta TNF-α, IL-1β da IL-6; (B) matakan furotin na hanji na TNF-α, IL-1β da IL-6. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 4–6).#p < 0.05 ko ##p < 0.01 ko ###p < 0.001 vs ƙungiyar sarrafawa (Con); *p < 0.05 ko **p < 0.01 vs ƙungiyar DSS.
Ciwon ciki yana da matuƙar muhimmanci a cikin cututtukan UC.24 Domin bincika ko BLG yana daidaita ƙwayoyin cuta na hanji na berayen da DSS ta yi wa magani, an yi jerin 16S rRNA don nazarin al'ummar ƙwayoyin cuta na abubuwan da ke cikin hanji. Zane-zanen Venn ya nuna cewa ƙungiyoyi uku suna da OTU 385. A lokaci guda, kowace ƙungiya tana da OTU na musamman (Hoto na 3A). Bugu da ƙari, ma'aunin Chao1 da ma'aunin Shannon da aka nuna a Hoto na 3B da C sun nuna cewa bambancin al'umma na ƙwayoyin cuta na hanji ya ragu a cikin berayen da aka yi wa magani na BLG, saboda ma'aunin Shannon ya ragu sosai a cikin ƙungiyar da aka yi wa magani na BLG. An yi amfani da babban binciken sassan (PCA) da babban nazarin daidaitawa (PCoA) don tantance tsarin rukuni tsakanin ƙungiyoyi uku kuma sun nuna cewa tsarin al'umma na berayen da DSS ta yi wa magani ya rabu a fili bayan maganin BLG (Hoto na 3D da E). Waɗannan bayanai sun nuna cewa maganin BLG ya shafi tsarin al'umma na berayen da ke fama da ciwon ciki da DSS ta haifar.
Siffa ta 3 BLG tana canza bambancin ƙwayoyin cuta na hanji a cikin beraye masu fama da cutar colitis da DSS ke haifarwa.(A) Zane-zanen Venn na OTU, (B) Ma'aunin Chao1, (C) Ma'aunin wadatar Shannon, (D) Tsarin maki na Babban Binciken Sassan (PCA) na OTU, (E) Ma'aunin OTU Babban Tsarin Binciken (PCoA) Hoto. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 6).**p < 0.01 vs ƙungiyar DSS.
Domin tantance takamaiman canje-canje a cikin ƙwayoyin cuta na hanji, mun yi nazarin abubuwan da ke cikin ƙwayoyin cuta na hanji a duk matakan taxonomic. Kamar yadda aka nuna a Hoto na 4A, manyan ƙwayoyin cuta a cikin dukkan ƙungiyoyi sune Firmicutes da Bacteroidetes, sai kuma Verrucomirobia. Yawan adadin ƙwayoyin cuta na Firmicutes da Firmicutes/Bacteroides ya ƙaru sosai a cikin al'ummomin ƙwayoyin cuta na berayen da aka yi wa magani da DSS idan aka kwatanta da berayen da aka sarrafa, kuma waɗannan canje-canjen sun koma baya sosai bayan maganin BLG. Musamman ma, maganin BLG ya ƙara yawan ƙwayoyin cuta na Verrucobacterium a cikin najasar berayen da ke fama da cutar colitis da DSS ta haifar. A matakin gida, al'ummomin ƙwayoyin cuta na hanji suna ƙarƙashin Lachnospiriaceae, Muribaculaceae, Akkermansiaceae, Ruminococcaceae da Prevotellaceae (Hoto na 4B). Idan aka kwatanta da ƙungiyar DSS, raguwar BLG ya ƙara yawan Akkermansiaceae, amma ya rage yawan Lachnospiraceae da Ruminococcaceae. Abin lura shi ne, a matakin jinsi, najasar Lachnospira_NK4A136_group, Akkermansia da Prevotellaceae_UCG-001 sun mamaye ƙwayoyin cuta (Hoto na 4C). Wannan binciken ya kuma nuna cewa maganin BLG ya canza rashin daidaiton ƙwayoyin cuta ta hanyar mayar da martani ga ƙalubalen DSS, wanda ke nuna raguwar ƙwayoyin cuta na Eubacterium_xylanophilum_group, Ruminococcaceae_UCG-014, Intestinimonas da Oscillibacter, da kuma ƙaruwar ƙwayoyin cuta na Akkermansia da Prevotellaceae_UCG-001.
Hoto na 4 BLG yana canza yawan ƙwayoyin cuta na hanji a cikin beraye masu fama da cutar colitis da DSS ke haifarwa.(A) Yawan ƙwayoyin cuta na hanji a matakin phylum; (B) Yawan ƙwayoyin cuta na hanji a matakin iyali; (C) Yawan ƙwayoyin cuta na hanji a matakin nau'in halittar. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 6).#p < 0.05 ko ###p < 0.001 vs ƙungiyar kulawa (Con); *p < 0.05 ko **p < 0.01 ko ***p < 0.001 vs ƙungiyar DSS.
Idan aka yi la'akari da cewa gajerun kitsen acid (SCFAs) sune manyan abubuwan da ke cikin Akkermansia da Prevotellaceae_UCG-001, yayin da acetate, propionate da butyrate sune mafi yawan SCFAs a cikin lumen hanji, har yanzu muna cikin bincikenmu. Kamar yadda aka nuna a Hoto na 5, yawan fecal acetate, propionate, da butyrate ya ragu sosai a cikin rukunin da DSS ta yi wa magani, yayin da maganin BLG zai iya rage wannan raguwar sosai.
Hoto na 5. BLG yana ƙara yawan SCFAs a cikin najasar beraye masu fama da cutar colitis da DSS ke haifarwa.(A) Yawan sinadarin acetic acid a cikin najasa; (B) yawan sinadarin propionic acid a cikin najasa; (C) yawan sinadarin butyric acid a cikin najasa. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 6).#p < 0.05 ko ##p < 0.01 vs ƙungiyar sarrafawa (Con); *p < 0.05 ko **p < 0.01 vs ƙungiyar DSS.
Mun ƙara ƙididdige ma'aunin haɗin Pearson tsakanin bambancin matakin SCFA na jinsi da na hanji microbiota. Kamar yadda aka nuna a Hoto na 6, Akkermansia yana da alaƙa mai kyau da samar da propionic acid (Pearson = 0.4866) da butyric acid (Pearson = 0.6192). Sabanin haka, duka Enteromonas da Oscillobacter suna da alaƙa mara kyau da samar da acetate, tare da ma'aunin Pearson na 0.4709 da 0.5104, bi da bi. Haka kuma, Ruminokoccaceae_UCG-014 yana da alaƙa mara kyau da samar da propionic acid (Pearson = 0.4508) da butyric acid (Pearson = 0.5842), bi da bi.
Hoto na 6 Binciken alaƙar Pearson tsakanin bambance-bambancen SCFAs da ƙwayoyin cuta na hanji.(A) Enteromonas tare da acetic acid; (B) Concussion bacillus tare da acetic acid; (C) Akkermansia vs propionic acid; (D) Ruminococcus_UCG-014 tare da propionic acid; (E) Akkermansia tare da butyric acid; (F) ) Ruminococcus _UCG-014 tare da butyric acid.
Peptide-1 mai kama da Glucagon (GLP-1) samfurin proglucagon (Gcg) ne na musamman bayan fassarar ƙwayoyin halitta tare da kaddarorin hana kumburi.28 Kamar yadda aka nuna a Hoto na 7, DSS ta haifar da raguwa mai yawa a cikin bayyanar mRNA na Gcg. Maganin colon da BLG na iya rage raguwar Gcg da DSS ta haifar idan aka kwatanta da ƙungiyar kulawa (Hoto na 7A). A lokaci guda, matakin GLP-1 a cikin jini ya ragu sosai a cikin ƙungiyar da DSS ta yi wa magani, kuma maganin BLG zai iya hana wannan raguwar sosai (Hoto na 7B). Tunda gajerun kitse na iya ƙarfafa fitar da GLP-1 ta hanyar kunna mai karɓar G-protein 43 (GRP43) da mai karɓar G-protein 41 (GRP41), mun kuma bincika GPR41 da GRP43 a cikin hanjin beraye na colitis kuma mun gano cewa bayyanar mRNA na colonic na GRP43 da GPR41 ya ragu sosai bayan ƙalubalen DSS, kuma maganin BLG zai iya ceton waɗannan raguwar yadda ya kamata (Hoto na 7C da D).
Hoto na 7 BLG yana ƙara matakan GLP-1 a cikin jini da bayyanar Gcg, GPR41 da GRP43 mRNA a cikin beraye da aka yi wa magani da DSS.(A) bayyanar Gcg mRNA a cikin kyallen hanji; (B) matakin GLP-1 a cikin jini; (C) bayyanar GPR41 mRNA a cikin kyallen hanji; (D) bayyanar GPR43 mRNA a cikin kyallen hanji. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 5–6).#p < 0.05 ko ##p < 0.01 vs ƙungiyar sarrafawa (Con); *p < 0.05 vs ƙungiyar DSS.
Tunda maganin BLG zai iya ƙara matakan GLP-1 a cikin jini, bayyanar mRNA na hanji, da matakan SCFA na najasa a cikin berayen da DSS ta yi wa magani, mun ƙara bincika acetate, propionate, da butyrate da kuma daga beraye masu sarrafawa (F-Con), DSS colitis (F-Con) -DSS) da beraye masu fama da cutar BLG (F-BLG) bayan fitowar GLP-1 daga manyan ƙwayoyin epithelial na hanji. Kamar yadda aka nuna a Hoto na 8A, ƙwayoyin epithelial na hanji na farko da aka yi wa magani da 2 mM acetic acid, propionic acid, da butyric acid, bi da bi, sun ƙarfafa sakin GLP-1 sosai, daidai da binciken da aka yi a baya.29,30 Haka kuma, duk F-Con, F-DSS, da F-BLG (daidai da 0.25 g na najasa) sun ƙarfafa sakin GLP-1 sosai daga manyan ƙwayoyin epithelial na hanji na hanji. Abin lura shi ne, adadin GLP-1 da ƙwayoyin epithelial na hanji na farko da F-DSS ta yi wa magani ya yi ƙasa da na F-Con da F-BLG ta yi wa magani Kwayoyin halittar hanji na beraye. (Hoto na 8B). Waɗannan bayanai sun nuna cewa maganin BLG ya dawo da samar da GLP-1 daga SCFA a cikin hanji sosai.
Siffa ta 8 SCFA da BLG ta samo daga SLF tana ƙarfafa sakin GLP-1 daga ƙwayoyin epithelial na hanji.(A) Acetic acid, propionic acid, da butyric acid sun ƙarfafa sakin GLP-1 daga ƙwayoyin epithelial na hanji na hanji; (B) cirewar najasa F-Con, F-DSS da F-BLG waɗanda aka ƙarfafa su daga ƙwayoyin epithelial na hanji na hanji Adadin GLP-1 da aka saki. An sanya adadin ƙwayoyin epithelial na hanji a cikin kwano na petri na gilashi kuma an yi musu magani da acetic acid 2 mM, propionic acid, butyric acid, da kuma cirewar najasa F-Con, F-DSS, da F-BLG (daidai da najasa 0.25 g), bi da bi. Awa 2 a zafin jiki na 37°C, 5% na CO2, bi da bi. ELISA ta gano adadin GLP-1 da aka saki daga manyan ƙwayoyin epithelial na hanji. An gabatar da bayanai a matsayin matsakaicin ± SEM (n = 3).#p < 0.05 ko ##p < 0.01 vs. blank ko F-Con; *p < 0.05 vs. F-DSS.
Takaitattun bayanai: Ace, acetic acid; Pro, propionic acid; duk da haka, butyric acid; F-Con, cirewar najasa daga beraye masu sarrafawa; F-DSS, cirewar najasa daga beraye masu ciwon ciki; F-BLG, daga cikin hanjin da aka yi wa magani da BLG. Cirewar najasa daga beraye masu kumburi.
Hukumar Lafiya ta Duniya ta sanya UC a matsayin cuta mai saurin kamuwa da cuta, kuma tana zama haɗari ga duniya; duk da haka, hanyoyin da suka dace don annabta, hanawa, da kuma magance cutar har yanzu suna da iyaka. Saboda haka, akwai buƙatar gaggawa don bincika da haɓaka sabbin dabarun magani masu aminci da tasiri ga UC. Shirye-shiryen maganin gargajiya na kasar Sin zaɓi ne mai kyau saboda an nuna cewa shirye-shiryen maganin gargajiya na kasar Sin da yawa suna da tasiri wajen magance UC a cikin al'ummar kasar Sin tsawon ƙarni, kuma duk ƙwayoyin halitta ne na halitta da kayan halitta waɗanda galibi ba su da lahani ga mutane da dabbobi.31,32 Wannan binciken yana da nufin neman ingantaccen shiri na maganin gargajiya na kasar Sin don maganin UC da kuma bincika tsarin aikinsa.BLG sanannen dabarar ganye ce ta kasar Sin da ake amfani da ita don magance mura.8,33 Aiki a dakin gwaje-gwajenmu da sauransu ya nuna cewa indigo, wani samfurin maganin gargajiya na kasar Sin da aka sarrafa daga kayan ƙasa iri ɗaya kamar BLG, yana nuna babban tasiri wajen maganin UC a cikin mutane da dabbobi.4,34 Duk da haka, tasirin hana colitis na BLG da tasirinsa Tsarin ba a fayyace shi ba. A cikin binciken da ake yi a yanzu, sakamakonmu ya nuna cewa BLG yana rage kumburin hanji da DSS ke haifarwa yadda ya kamata, wanda ke da alaƙa da daidaita ƙwayoyin cuta na hanji da dawo da ƙwayoyin cuta da aka samo daga hanji. Samar da GLP-1.
Sanannen abu ne cewa UC tana da alaƙa da sake dawowar lokaci tare da halaye na asibiti na yau da kullun, kamar rage nauyi, gudawa, zubar jini na dubura, da kuma lalacewar mucosa na hanji mai yawa.35 Don haka, an ba da ciwon colitis na yau da kullun ta hanyar ba da zagaye uku na 1.8% DSS na tsawon kwanaki biyar, sannan aka biyo baya kwana bakwai na shan ruwa. Kamar yadda aka nuna a Hoto na 1B, raguwar nauyi da maki na DAI sun nuna nasarar haifar da ciwon colitis na yau da kullun. Beraye a cikin rukunin da aka yi wa magani da BLG sun nuna murmurewa mai yawa daga rana ta 8, wanda ya bambanta sosai da rana ta 24. An kuma lura da irin waɗannan canje-canje a cikin maki na DAI, yana nuna ci gaba a cikin ci gaban ciwon colitis na asibiti. Dangane da raunin hanji da yanayin kumburi, tsawon hanji, lalacewar kyallen hanji, da bayyanar kwayoyin halitta da samar da cytokines masu kumburi TNF-α, IL-1β, da IL-6 a cikin kyallen hanji suma an inganta su sosai bayan maganin BLG. Ta hanyar haɗin gwiwa, waɗannan sakamakon sun nuna a fili cewa BLG yana da tasiri wajen magance ciwon colitis na yau da kullun a cikin beraye.
Ta yaya BLG ke yin tasirin maganin sa? Nazarce-nazarce da dama da aka yi a baya sun nuna cewa ƙwayoyin cuta na hanji suna taka muhimmiyar rawa a cikin cututtukan UC, kuma magungunan da aka yi amfani da su ta hanyar microbiome da microbiome sun fito a matsayin wata dabara mai kyau don maganin UC. A cikin wannan binciken, mun nuna cewa maganin BLG ya haifar da manyan canje-canje a cikin abun da ke cikin ƙwayoyin cuta na hanji, yana nuna cewa tasirin kariya na BLG akan colitis da DSS ke haifarwa yana da alaƙa da daidaita ƙwayoyin cuta na hanji. Wannan lura ya yi daidai da ra'ayin cewa sake tsara tsarin homeostasis na ƙwayoyin cuta na hanji muhimmin hanya ne don fahimtar ingancin shirye-shiryen TCM.36,37 Abin lura, Akkermansia ƙwayar cuta ce mai kama da Gram-negative kuma mai tsananin rashin lafiyar jiki wadda ke rayuwa a cikin laka na mucosa na hanji, wanda ke lalata mucins, yana samar da propionic acid, yana ƙarfafa bambance-bambancen ƙwayoyin cuta na goblet, kuma yana kula da mucosa. aikin amincin shinge.26 Bayanan asibiti da dabbobi da yawa sun nuna cewa Akkermansia yana da alaƙa sosai da mucosa mai lafiya,38 da kuma shan ta baki na Akkermansia spp. zai iya inganta kumburin mucosa sosai.39 Bayananmu na yanzu sun nuna cewa yawan Akkermansia yana ƙaruwa sosai bayan maganin BLG. Bugu da ƙari, Prevotellaceae_UCG-001 ƙwayar cuta ce da ke samar da SCFA.27 Bincike da yawa sun nuna cewa an gano Prevotellaceae_UCG-001 a cikin ƙarancin yawan dabbobi a cikin najasar dabbobi masu fama da cutar colitis.40,41 Bayananmu na yanzu sun kuma nuna cewa maganin BLG na iya ƙara yawan Prevotellaceae_UCG-001 a cikin hanjin beraye da aka yi wa magani da DSS. Sabanin haka, Oscillibacter ƙwayar cuta ce mai kama da mesophilic, mai kama da anaerobic.42 ta ba da rahoton cewa yawan Oscillibacter ya ƙaru sosai a cikin beraye na UC kuma yana da alaƙa mai kyau da matakan IL-6 da IL-1β da sakamakon cututtuka.43,44 Abin lura, maganin BLG ya rage yawan Oscillibacter a cikin najasar beraye da aka yi wa magani da DSS. Abin lura, waɗannan ƙwayoyin cuta da aka canza da BLG sune ƙwayoyin cuta mafi yawan samar da SCFA. Nazarin da yawa da suka gabata sun nuna yuwuwar tasirin amfani na SCFAs akan kumburin hanji da kuma kariyar ingancin epithelial na hanji.45,46 Bayananmu na yanzu sun kuma lura cewa yawan SCFA acetate, propionate, da butyrate a cikin najasar da DSS ta yi wa magani ya ƙaru sosai a cikin beraye da aka yi wa magani da BLG. Idan aka haɗa su, waɗannan binciken sun nuna a sarari cewa maganin BLG zai iya haɓaka ƙwayoyin cuta masu samar da SCFA da DSS ta haifar a cikin beraye masu fama da ciwon hanji na yau da kullun.
GLP-1 wani incretin ne da ake samarwa galibi a cikin ileum da hanji kuma yana taka muhimmiyar rawa wajen jinkirta fitar da ruwa daga ciki da kuma rage glucose na jini bayan an fara amfani da shi.47 Shaida ta nuna cewa dipeptidyl peptidase (DPP)-4, mai maganin GLP-1, da kuma maganin nanomedicine na GLP-1 na iya rage kumburin hanji a cikin beraye yadda ya kamata.48-51 Kamar yadda aka ruwaito a cikin binciken da suka gabata, yawan SCFA yana da alaƙa da matakan GLP-1 a cikin jini a cikin mutane da beraye. 52 Bayananmu na yanzu sun nuna cewa bayan maganin BLG, matakan GLP-1 a cikin jini da bayyanar mRNA na Gcg sun ƙaru sosai. Haka kuma, fitar da GLP-1 ya ƙaru sosai a cikin al'adun hanji bayan ƙarfafawa da cirewar najasa daga beraye da aka yi wa maganin colitis da BLG idan aka kwatanta da ƙarfafawa da cirewar najasa daga beraye da aka yi wa maganin colitis da DSS ta yi wa magani. Ta yaya SCFAs ke shafar sakin GLP-1?Gwen Tolhurst et al. An ruwaito cewa SCFA na iya ƙarfafa fitar da GLP-1 ta hanyar GRP43 da GPR41.29 Bayananmu na yanzu sun kuma nuna cewa maganin BLG yana ƙara yawan bayyanar mRNA na GRP43 da GPR41 a cikin hanjin beraye da aka yi wa magani da DSS. Waɗannan bayanan sun nuna cewa maganin BLG na iya dawo da samar da GLP-1 da SCFA ta haɓaka ta hanyar kunna GRP43 da GPR41.
BLG magani ne da ake amfani da shi ba tare da takardar likita ba (OTC) a China na dogon lokaci. Matsakaicin adadin BLG da ake yarda da shi a cikin beraye Kunming shine 80g/Kg, kuma ba a lura da guba mai tsanani ba.53 A halin yanzu, shawarar da aka bayar na BLG (ba tare da sukari ba) a cikin mutane shine 9-15 g/rana (sau 3 a rana). Bincikenmu ya nuna cewa BLG a 1g/Kg yana rage yawan ciwon hanji da DSS ke haifarwa a cikin beraye. Wannan kashi yana kusa da adadin BLG da ake amfani da shi a asibiti. Bincikenmu ya kuma gano cewa tsarin aikinsa yana da alaƙa, aƙalla a wani ɓangare, ta hanyar canje-canje a cikin ƙwayoyin cuta na hanji, musamman ƙwayoyin cuta masu samar da SCFA, kamar Akkermansia da Prevotellaceae_UCG-001, don dawo da samar da GLP-1 daga hanji. Waɗannan binciken sun nuna cewa BLG ya cancanci ƙarin la'akari a matsayin mai yuwuwar maganin ciwon hanji don maganin ciwon hanji na asibiti. Duk da haka, ainihin hanyar da yake daidaita ƙwayoyin cuta na hanji har yanzu ba a tabbatar da ita ba ta hanyar beraye masu ƙarancin ƙwayoyin cuta da dashen ƙwayoyin cuta na hanji.
Ace, acetic acid; but, butyric acid; BLG, pandan; DSS, dextran sodium sulfate; DAI, ma'aunin ayyukan cuta; DPP, dipeptidyl peptidase; FID, mai gano ionization na harshen wuta; F-Con, sarrafa cirewar najasa na beraye; F-DSS, cirewar najasa na berayen DSS colitis; F-BLG, cirewar najasa na berayen colitis da aka yi wa magani da BLG; GLP-1, glucagon-like peptide-1; Gcg, glucagon; gas chromatography, gas chromatography; GRP43, G protein-coupled receptor 43; GRP41, G protein-coupled receptor 41; H&E, hematoxylin-eosin; HBSS, Hanks' Balanced Gishiri Solution; OTC, OTC; PCA, babban nazarin sinadaran; PCoA, babban nazarin daidaitawa; Pro, propionic acid; SASP, sulfasalazine; SCFA, gajerun kitse mai kitse; maganin kasar Sin, maganin gargajiya na kasar Sin; UC, ulcerative colitis.
Kwamitin Ɗabi'ar Dabbobi na Cibiyar Kiwon Lafiya ta Jami'ar Peking ta Shenzhen-Hong Kong (Shenzhen, China) ya amince da dukkan ka'idojin gwaji bisa ga Ka'idojin Cibiyoyin da Dokokin Dabbobi (lambar ɗabi'a A2020157).
Duk marubuta sun ba da gudummawa mai mahimmanci ga tunani da ƙira, tattara bayanai, ko nazarin bayanai da fassara su; sun shiga cikin rubuta labarin ko yin bita mai mahimmanci kan mahimman abubuwan ilimi; sun amince su miƙa rubutun ga mujallar da ke yanzu; a ƙarshe sun amince da sigar don bugawa; Mai alhakin dukkan fannoni na aiki.
Gidauniyar Kimiyyar Halitta ta Ƙasa ta ƙasar Sin (81560676 da 81660479), aikin farko na Jami'ar Shenzhen (86000000210), Asusun Kwamitin Ƙirƙirar Kimiyya da Fasaha na Shenzhen (JCYJ20210324093810026), da Asusun Binciken Kimiyya da Fasaha na Lafiya na lardin Guangdong (A2020157 da A2020272), Magungunan Jami'ar Likitanci ta Guizhou Lardin Guizhou wanda Babban Dakin Gwaji (YWZJ2020-01) da Asibitin Shenzhen na Jami'ar Peking (JCYJ2018009) suka tallafa wa wannan aikin.
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