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Mkpụrụ ndụ Ban-Lan-Gen na-ebelata ọrịa afọ ọsịsa nke dextran sodium sulfate kpatara n'ime òké site n'ịgbanwe microbiota eriri afọ na iweghachi mmepụta eriri afọ SCFA Derived-GLP-1.
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 Mahadum Peking Shenzhen Hospital Ngalaba Ọgwụ, Shenzhen, People's Republic of China; 2Shenzhen University Health Science Center School of Pharmacy, Shenzhen, People's Republic of China; 3Guizhou Medical University Engineering Technology Research Center of Ethnic Medicine and Traditional Chinese Medicine Development and Application Ministry of Education, Guizhou Provincial Key Laboratory of Pharmacy, Guizhou Medical University, Guiyang, People's Republic of China; 4 Ngalaba Gastroenterology, Peking University Shenzhen Hospital, Shenzhen, People's Republic of China; 5 School of Pharmacy, Guizhou Medical University, State Key Laboratory of Medicine Plant Function and Application, Guiyang; Ngalaba Ọgwụ Ụlọ Nyocha 6, Ụlọ Ọgwụ Shenzhen nke Mahadum Peking, Shenzhen, China [email protected] Shen Xiangchun, Ụlọ Akwụkwọ Ọgwụ, Mahadum Ahụike Guizhou, Guizhou, Ndị Mmadụ nke China, 550004, Email [email protected] Ebumnuche: Ọgwụgwọ dabere na GLP-1 bụ nhọrọ ọgwụgwọ ọhụrụ maka ọrịa eriri afọ mkpali. Mkpụrụ ndụ Ban-Lan-Gen (BLG) bụ usoro TCM a ma ama nke na-egosi ọrụ mgbochi mkpali nwere ike ịgwọ ọrịa dị iche iche nke ọrịa mkpali. Agbanyeghị, mmetụta mgbochi mkpali ya na ọrịa afọ na usoro ọrụ ya ka edoghị anya. ỤZỌ: Iji guzobe colitis nlọghachi azụ nke dextran sodium sulfate (DSS) kpatara na òké. E mere ihe ngosi ọrụ ọrịa, ihe nrịbama histological nke mmerụ ahụ, na ọkwa cytokine proinflammatory iji nyochaa mmetụta nchebe nke BLG. Mmetụta nke BLG na microbiota eriri afọ na eriri afọ bụ ihe e ji mara ọkwa GLP-1 serum na ngosipụta Gcg colonic, GPR41, na GRP43, nhazi microbiota eriri afọ, ọkwa SCFAs fecal, na Mwepụta GLP-1 site na mkpụrụ ndụ epithelial nke oke bekee bụ isi mmepụta GLP-1 sitere na SCFA. Nsonaazụ: Ọgwụgwọ BLG belatara nke ukwuu mbelata ibu ahụ, DAI, mkpụmkpụ eriri afọ, mmebi anụ ahụ eriri afọ, na ọkwa cytokine proinflammatory nke TNF-α, IL-1β, na IL-6 na anụ ahụ eriri afọ. Na mgbakwunye, ọgwụgwọ BLG nwere ike iweghachi oke ngosipụta Gcg, GPR41 na GRP43 na ọkwa GLP-1 nke eriri afọ na oke eriri afọ, yana site na ịbawanye nje bacteria na-emepụta SCFA dị ka Akkermansia na Prevotellaceae_UCG-001, na ibelata ụba nke nje bacteria dị ka Eubacterium_xylanophilum_group, Ruminokoccaceae_UCG-014, Intestinimonas na Oscillibacter. Na mgbakwunye, ọgwụgwọ BLG nwere ike ime ka ọkwa SCFA dịkwuo elu nke ukwuu na nsị nke òké eriri afọ. N'otu oge ahụ, nnwale in vitro gosikwara na ihe nsị nke òké ejiri BLG gwọọ nwere ike ịkpalite obere mkpụrụ ndụ epithelial Murine nke eriri afọ na-ewepụta GLP-1 nke ukwuu. Mmechi: Nchọpụta ndị a na-egosi na BLG nwere mmetụta mgbochi afọ ntachi. BLG nwere ike ịmepụta ya dị ka ọgwụgwọ, ma ọ dịkarịa ala site n'ịgbanwe microbiota eriri afọ na iweghachi mmepụta GLP-1 nke SCFA sitere na eriri afọ Ọgwụ ndị na-ekwe nkwa maka afọ ntachi na-aga n'ihu ogologo oge. Okwu ndị dị mkpa: colitis, granules Ban-Lan-Gen, microbiota eriri afọ, obere abụba acids, GLP-1
Ulcerative colitis (UC) bụ ọrịa na-egbu mgbu ogologo oge nke eriri afọ na rectum nke a na-akpọ afọ ọsịsa ugboro ugboro, mgbu afọ, mbelata ibu, na nsị ọbara mucopurulent.1 N'oge na-adịbeghị anya, mmụba nke UC na-amụba na mba ndị na-adịghị enwe mmetụta mbụ, gụnyere China, na-abawanye ewu ewu nke ụdị ndụ ndị ọdịda anyanwụ.2 Mmụba a na-akpata nnukwu nsogbu maka ahụike ọha na eze ma nwee nnukwu mmetụta maka ikike ndị ọrịa ịrụ ọrụ na ịdị mma nke ndụ. N'ụzọ dị mkpa, ọrịa nke UC ka edoghị anya nke ọma, mana a na-anabatakarị na mkpụrụ ndụ ihe nketa, ihe ndị metụtara gburugburu ebe obibi, microbiota eriri afọ, na sistemu ahụ ji alụso ọrịa ọgụ niile na-enye aka na mmepe nke UC.3 Ọbụna ugbu a, enweghị ọgwụgwọ maka UC, ebumnuche nke ọgwụgwọ bụ ijikwa mgbaàmà ahụike, ịkpalite ma nọgide na-enwe mgbaghara, kwalite ọgwụgwọ mucosal, na ibelata nlọghachi. Ọgwụgwọ ochie gụnyere aminosalicylates, corticosteroids, immunosuppressants, na biologics. Agbanyeghị, ọgwụ ndị a enweghị ike iru mmetụta achọrọ n'ihi mmetụta dị iche iche ha nwere.4 N'oge na-adịbeghị anya, ọtụtụ ọmụmụ ihe egosila na ọgwụ ọdịnala China (TCM) egosila nnukwu ike n'inyere aka belata UC na obere nsí, na-egosi na Mmepe nke ọgwụgwọ TCM ọhụrụ bụ atụmatụ ọgwụgwọ na-ekwe nkwa maka UC.5-7​ ...
Banlangen Granules (BLG) bụ nkwadebe ọgwụ ọdịnala nke ndị China nke e ji mmiri wepụ ihe na mgbọrọgwụ Banlangen.8 Na mgbakwunye na irè ya na nje virus, BLG na-egosipụta ọrụ mgbochi mkpali n'ịgwọ ọrịa dị iche iche nke ọrịa mkpali.9,10 Na mgbakwunye, a kewapụrụ glucosinolates (R,S-goitrin, progoitrin, epiprorubin na glucoside ma chọpụta ya site na ihe ndị sitere na mmiri Radix isatidis) na nucleosides (hypoxanthine, adenosine, uridine na guanosine) na indigo alkaloids dị ka indigo na indirubin.11,12 Ọmụmụ ihe ndị gara aga edekọwo nke ọma na ihe ndị mejupụtara adenosine, uridine na indirubin na-egosipụta mmetụta dị ike nke mgbochi colitis na ụdị anụmanụ dị iche iche nke colitis.13-17 Agbanyeghị, emebeghị ọmụmụ ihe ọ bụla dabere na ihe akaebe iji nyochaa irè nke BLG na colitis. Na ọmụmụ ihe a, anyị nyochara mmetụta nchebe nke BLG na dextran sodium sulfate (DSS) nke kpatara ọrịa mgbu na-adịghị ala ala na oke C57BL/6 wee chọta ya. na inye BLG ọnụ belatara nke ukwuu ihe DSS kpatara site n'iji eriri afọ na-alọghachi azụ n'ime òké. Mbufụt, usoro nhazi ya jikọtara ya na mgbanwe nke microbiota eriri afọ na mweghachi nke mmepụta glucagon-like peptide-1 (GLP-1) sitere na eriri afọ.
A zụtara mkpụrụ ọgwụ BLG (enweghị shuga, nke NMPA kwadoro Z11020357; Beijing Tongrentang Technology Development Co., Ltd., Beijing, China; nọmba ogbe: 20110966) n'ụlọ ahịa ọgwụ. A zụtara DSS (Ọnụọgụ molekul: 36,000–50,000 Daltons) n'aka MP Biologicals (Santa Ana, USA). A zụtara Sulfasalazine (SASP) (≥ 98% ịdị ọcha), hematoxylin na eosin n'aka Sigma-Aldrich (St. Louis, MO, USA). A zụtara ngwa nnwale Elisa nke Mouse TNF-α, IL-1β na IL-6 luminex site na sistemụ R&D (Minneapolis, MN, USA). A zụtara acetic acid, propionic acid, na butyric acid site na Aladdin Industries (Shanghai, China). A zụtara 2-Ethylbutyric acid site na Merck KGaA (Darmstadt, Germany).
E zụtara òké nwoke dị izu 6-8 C57BL/6 (ibu ahụ ya dị 18-22 g) na Beijing Wetahe Laboratory Animal Technology Co., Ltd. (Beijing, China) ma debe ha n'ebe okpomọkụ dị 22 ± 2 °C yana okirikiri ọkụ/ọchịchịrị awa 12. E nyere òké nri òké nkịtị, ha nwere ike ịnweta mmiri ọṅụṅụ n'efu ruo otu izu iji mee ka ha dabara na gburugburu ebe obibi ọhụrụ ahụ. E kewara òké ndị ahụ n'ime otu anọ n'enweghị usoro: otu njikwa, otu ụdị DSS, otu agwọ SASP (200 mg/kg, ọnụ) na otu agwọ BLG (1 g/kg, ọnụ). Dịka egosiri na Foto 1A, dịka ọmụmụ ihe anyị gara aga si dị, a kpalitere ọrịa afọ ọsịsa na-adịghị ala ala n'ime òké site na usoro atọ nke 1.8% DSS maka ụbọchị ise, wee soro mmiri a gwakọtara agwakọta ruo ụbọchị asaa, dịka ọmụmụ ihe anyị gara aga si dị.18 E jiri SASP na BLG gwọọ òké ndị nọ n'ìgwè agwọ SASP na BLG, n'otu n'otu, kwa ụbọchị malite na ụbọchị 0. Dịka nnwale mbụ si dị, a na-agwọ òké ndị a na SASP na BLG, kwa ụbọchị malite na ụbọchị 0. Dịka nnwale mbụ si dị, dose nke E tinyere BLG na 1 g/kg. Ka ọ dị ugbu a, e tinyere dose SASP na 200 mg/Kg dịka akwụkwọ si kwuo.4 Ndị otu njikwa na ndị otu DSS natara otu olu mmiri ahụ n'oge nnwale ahụ.
Foto nke 1 BLG na-ebelata ọrịa afọ ntachi nke DSS kpatara n'ime òké.(A) Nhazi nnwale nke ọrịa afọ ntachi na ọgwụgwọ na-adịghị ala ala, (B) mgbanwe ibu ahụ, (C) akara ọrụ ọrịa (DAI), (D) ogologo eriri afọ, (E) onyonyo nnọchi anya nke eriri afọ, (F) H&E staining Colon (magnification, ×100) na (G) akara histological. A na-egosi data dị ka nkezi ± SEM (n = 6).##p < 0.01 ma ọ bụ ###p < 0.001 vs otu njikwa (Con); *p < 0.05 ma ọ bụ **p < 0.01 ma ọ bụ ***p < 0.001 vs otu DSS.
A na-edekọ ịdị arọ ahụ, otú o siri dị n'oche, na ọbara ọgbụgba n'ime imi kwa ụbọchị. E kpebiri ihe ngosi ọrụ ọrịa (DAI) site na ijikọta ọtụtụ nha ahụ, otú o si dị n'oche, na ọbara ọgbụgba n'ime imi dịka akọwara na mbụ.19 Ná ngwụcha nnwale ahụ, e gburu òké niile ma chịkọta ọbara, nsị na eriri afọ maka nnwale ndị ọzọ.
A na-etinye anụ ahụ eriri afọ nke ọma ma tinye ya na paraffin. E mepụtara akụkụ 5-micron ma tee ya agba na hematoxylin-eosin (H&E), wee kpuo ìsì ma nye akara dịka akọwara na mbụ.19
E jiri ihe Trizol reagent (Invitrogen, Carlsbad, CA) wepụta RNA zuru oke nke anụ ahụ eriri afọ, wee wepụta cDNA na reverse transcriptase (TaKaRa, Kusatsu, Shiga, Japan). Ejiri usoro PCR oge a na SYBR Green Master (Roche, Basel, Switzerland) mee PCR ọnụọgụgụ. E mere ka mkpụrụ ndụ ihe nketa Target bụrụ β-actin ma nyochaa data site na iji usoro 2-ΔΔCT. E gosiri usoro primer mkpụrụ ndụ ihe nketa na Tebụl 1.
E mere mwepụta na mmepe nke mkpụrụ ndụ epithelial nke oke bekee dịka akọwara na mbụ.20 N'ụzọ dị mkpirikpi, a na-ebu ụzọ chụpụ eriri afọ nke òké dị izu 6-8 mgbe a chụsịrị ha site na nkwarụ nke cervical, wee mepee ya ogologo oge, jiri Hanks Balanced Salt Solution (HBSS, na-enweghị calcium na magnesium) gwọọ ha ma bepụ ha n'ime obere iberibe 0.5-1 mm. Mgbe nke ahụ gasịrị, a gbari anụ ahụ na 0.4 mg/mL collagenase XI (Sigma, Poole, UK) na DMEM medium n'efu ma tinye centrifuge na 300 xg maka nkeji ise na okpomọkụ ụlọ. Tinyeghachi pellet ahụ na DMEM medium (agbakwunyere na 10% fetal bovine serum, 100 Units/mL penicillin, na 100 µg/mL streptomycin) na 37 °C ma gafere naịlọn mesh (nha oghere ~250 µm). E tinyere mkpụrụ ndụ epithelial colonic n'ime efere iko ma tinye acetic acid, propionic acid, butyric acid, na ihe e si na nsị òké wepụta ruo awa abụọ na okpomọkụ 37°C, 5% CO2.
E jiri PBS mee ka anụ ahụ eriri afọ yie otu, a chọpụtakwara ọkwa nke cytokines IL-6, TNF-α na IL-1β na anụ ahụ eriri afọ site na iji ihe nyocha luminex ELISA (usoro R&D, Minneapolis, MN, USA). N'otu aka ahụ, ejiri ngwa ELISA (Bioswamp, Wuhan, China) chọpụta ọkwa GLP-1 na serum na culture medium nke mkpụrụ ndụ epithelial murine colonic mbụ dịka ntuziaka onye nrụpụta si dị.
E ji ngwa mwepụta DNA (Tiangen, China) wepụta DNA zuru oke site na nsị. A tụrụ ogo na ọnụọgụ DNA na oke nke 260 nm/280 nm na 260 nm/230 nm, n'otu n'otu. Mgbe nke a gasịrị, site na iji DNA ọ bụla ewepụtara dị ka ihe nlereanya, ejiri praịma 338F (ACTCCTACGGGAGGCAGCAG) na 806R (GGACTACHVGGGTWTCTAAT) mee ka mpaghara V3-V4 nke mkpụrụ ndụ ihe nketa 16S rRNA dịkwuo elu na mpaghara dị iche iche. E jiri QIAquick Gel Extraction Kit (QIAGEN, Germany) sachaa ngwaahịa PCR, nke a na-enyocha site na PCR oge, ma jiri usoro IlluminaMiseq PE300 sequencing (Illumina Inc., CA, USA) mee usoro. Maka nyocha bioinformatics, emere nhazi data na-eso usoro ndị akọpụtara na mbụ.21,22 Na nkenke, jiri Cutadapt (V1.9.1) mee nzacha faịlụ express raw. E ji otu chịkọta OTUs. UPARSE (ụdị 7.0.1001) nwere myirịta nke 97%, e jikwa UCHIME wepụ usoro chimeric. E jiri RDP classifier (http://rdp.cme.msu.edu/) mee nyocha na nhazi nkewa nke obodo dabere na nchekwa data mkpụrụ ndụ ihe nketa SILVA ribosomal RNA.
A tụrụ ọkwa nke abụba dị mkpụmkpụ (acetic acid, propionic acid, na butyric acid) dịka Tao na ndị otu ya si kọwaa na mbụ, yana mgbanwe ụfọdụ.23 N'ụzọ dị mkpirikpi, e buru ụzọ kwụnye 100 mg nke nsị n'ime 0.4 mL nke mmiri e gwepịara egwepịa, wee soro 0.1 mL nke 50% sulfuric acid na 0.5 mL nke 2-ethylbutyric acid (ọkọlọtọ dị n'ime), wee mee ka ọ dị ka otu ma kpoo ọkụ na 4°C. Centrifuge na 12,000 rpm maka nkeji iri na ise na C. E jiri 0.5 mL nke ether wepụta ihe dị n'elu ya ma gbanye ya n'ime GC maka nyocha. Maka nyocha gas chromatography (GC), e jiri GC-2010 Plus gas chromatograph (Shimadzu, Inc.) nke nwere ihe nchọpụta ionization ọkụ (FID) nyochaa ihe nlele. E jiri kọlụm ZKAT-624, 30 m × 0.53 mm × 0.3 μm (Lanzhou Zhongke Antai Analytical Technology Co., Ltd., China nweta nkewa ahụ. E jiri ngwanrọ ngwọta GC (Shimadzu, Inc.) nweta data. Oke nkewa ahụ bụ 10:1, gas na-ebu ya bụ nitrogen, ọnụego mmiri ahụ bụkwa 6 mL/min. Olu nke ịgba ya bụ 1 μL. Okpomọkụ nke injector na detector bụ 300°C. E jidere okpomọkụ oven na 140°C maka nkeji 13.5, wee mụbaa ruo 250°C na ọnụego nke 120°C/min; a na-ejide okpomọkụ ahụ ruo nkeji 5.
A na-egosi data dị ka nkezi ± njehie ọkọlọtọ nke nkezi (SEM). E jiri otu ụzọ ANOVA nyochaa mkpa data wee soro ule Duncan nke ọtụtụ oke. E jiri ngwanrọ GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) mee ihe maka ngụkọ niile ma a na-ewere p < 0.05 dị ka ihe dị mkpa na ọnụọgụgụ.
A maara nke ọma na UC bụ ọrịa colitis na-aga n'ihu nke na-enwe nnukwu mgbu afọ, afọ ọsịsa na ọbara ọgbụgba. Ya mere, e guzobere colitis na-aga n'ihu nke DSS kpatara n'ime òké iji nyochaa irè nke BLG (Foto 1A). Ma e jiri ya tụnyere otu njikwa, òké dị na otu nlereanya DSS belatara ibu ahụ nke ukwuu ma nwee DAI dị elu, mgbanwe ndị a agbanwekwara nke ukwuu mgbe ụbọchị 24 nke ọgwụgwọ BLG gasịrị (Foto 1B na C). Mkpụmkpụ colon bụ ihe dị mkpa nke UC. Dịka egosiri na Foto 1D na E, ogologo eriri afọ nke òké natara DSS dị mkpụmkpụ nke ukwuu, mana ọgwụgwọ BLG belatara. N'ikpeazụ, emere nyocha histopathological iji nyochaa mbufụt eriri afọ. Foto ndị e sere na akara ọrịa H&E gosiri na nchịkwa DSS mebiri usoro nhazi eriri afọ nke ukwuu ma butere mbibi crypt, ebe ọgwụgwọ BLG belatara mbibi crypt na akara ọrịa (Foto 1F na G). N'ụzọ dị mkpa, mmetụta nchebe nke BLG na dose nke 1 g/Kg yiri nke SASP na dose nke 200. mg/Kg. N'ozuzu, nchọpụta ndị a na-egosi na BLG dị irè n'ibelata oke ọrịa colitis na-adịghị ala ala nke DSS kpatara n'ime òké.
TNF-α, IL-1β na IL-6 bụ ihe dị mkpa na-egosi mbufụt nke eriri afọ. Dịka egosiri na Foto 2A, DSS butere mmụba dị ukwuu na ngosipụta mkpụrụ ndụ ihe nketa nke TNF-α, IL-1β na IL-6 na eriri afọ ma e jiri ya tụnyere otu njikwa. Nchịkwa nke BLG nwere ike ịgbanwe mgbanwe ndị a nke DSS na-akpata nke ukwuu. Na-esote, anyị jiri ELISA chọpụta ọkwa nke cytokines mkpali TNF-α, IL-1β, na IL-6 na anụ ahụ eriri afọ. Nsonaazụ ahụ gosikwara na ọkwa eriri afọ nke TNF-α, IL-1β, na IL-6 mụbara nke ukwuu na òké ejiri DSS gwọọ, ebe ọgwụgwọ BLG belatara mmụba ndị a (Foto 2B).
Foto nke 2 BLG na-egbochi ngosipụta mkpụrụ ndụ ihe nketa na mmepụta nke cytokines proinflammatory TNF-α, IL-1β na IL-6 n'ime eriri afọ nke òké ndị DSS gwọrọ.(A) Ngosipụta mkpụrụ ndụ ihe nketa nke TNF-α, IL-1β na IL-6; (B) ọkwa protein colonic nke TNF-α, IL-1β na IL-6. A na-egosi data dị ka nkezi ± SEM (n = 4–6).#p < 0.05 ma ọ bụ ##p < 0.01 ma ọ bụ ###p < 0.001 vs otu njikwa (Con); *p < 0.05 ma ọ bụ **p < 0.01 vs otu DSS.
Ọrịa dysbiosis n'ime afọ dị oke mkpa na mmepe nke UC.24 Iji nyochaa ma BLG na-agbanwe microbiota eriri afọ nke òké ndị DSS gwọrọ, e mere usoro 16S rRNA iji nyochaa obodo nje bacteria nke ọdịnaya eriri afọ. Ihe osise Venn na-egosi na otu atọ ahụ nwere OTU 385. N'otu oge ahụ, otu ọ bụla nwere OTU pụrụ iche (Fig. 3A). Ọzọkwa, ihe ndekọ Chao1 na ihe ndekọ Shannon egosiri na Foto 3B na C gosiri na ụdị dị iche iche nke microbiota eriri afọ belatara na òké ndị BLG gwọrọ, ebe ihe ndekọ Shannon belatara nke ukwuu na otu ndị BLG gwọrọ. Ejiri nyocha akụkụ bụ isi (PCA) na nyocha nhazi isi (PCoA) chọpụta usoro nhazi n'etiti otu atọ ahụ ma gosi na e kewapụrụ usoro obodo nke òké ndị DSS gwọrọ nke ọma mgbe ọgwụgwọ BLG gasịrị (Foto 3D na E). Data ndị a na-egosi na ọgwụgwọ BLG metụtara usoro obodo nke òké ndị nwere colitis DSS gwọrọ nke ukwuu.
Foto nke 3 BLG na-agbanwe ụdị dị iche iche nke nje bacteria n'ime oke nwere ọrịa colitis nke DSS kpatara.(A) Eserese Venn nke OTU, (B) Ndekọ Chao1, (C) Ndekọ richness nke Shannon, (D) Atụmatụ akara nke Isi Nyocha (PCA) nke OTU, (E) Akara OTU Isi Nhazi Nchịkọta (PCoA) Foto. E gosipụtara data dị ka nkezi ± SEM (n = 6).**p < 0.01 vs otu DSS.
Iji chọpụta mgbanwe ụfọdụ na nje bacteria nsi, anyị nyochachara ihe mejupụtara nje bacteria n'afọ na ọkwa taxonomic niile. Dịka egosiri na Foto 4A, isi phyla n'ime otu niile bụ Firmicutes na Bacteroidetes, nke Verrucomirobia sochiri. Ọnụọgụ Firmicutes na Firmicutes/Bacteroides mụbara nke ukwuu na mpaghara nje bacteria nke òké ndị DSS gwọrọ ma e jiri ya tụnyere òké ndị na-achịkwa, mgbanwe ndị a agbanwekwara nke ukwuu mgbe ọgwụgwọ BLG gasịrị. Karịsịa, ọgwụgwọ BLG mụbara nke ukwuu nke Verrucobacterium n'ime nsị òké nwere colitis nke DSS kpatara. N'ezinụlọ, Lachnospiriaceae, Muribaculaceae, Akkermansiaceae, Ruminococcaceae na Prevotellaceae bi na mpaghara nje bacteria (Fig. 4B). Ma e jiri ya tụnyere otu DSS, mbelata nke BLG mụbara ụba nke Akkermansiaceae, mana belatara ụba nke Lachnospiraceae na Ruminococcaceae. Nke a pụtara ìhè, n'ọkwa mkpụrụ ndụ ihe nketa, nsị Lachnospira_NK4A136_group, Akkermansia na Prevotellaceae_UCG-001 bi na nje bacteria (Foto 4C). Nchọpụta a gosikwara na ọgwụgwọ BLG gbanwere enweghị nha nhata nke nje bacteria n'ụzọ dị irè na nzaghachi maka ihe ịma aka DSS, nke a na-egosi mbelata na Eubacterium_xylanophilum_group, Ruminokoccaceae_UCG-014, Intestinimonas na Oscillibacter, na mmụba na Akkermansia na Prevotellaceae_UCG-001.
Foto nke 4 BLG na-agbanwe oke nje bacteria n'ime oke colitis nke DSS kpatara.(A) Oke nje bacteria n'ime oke nje bacteria n'ime oke nje bacteria; (B) Oke nje bacteria n'ime oke nje bacteria n'ime oke nje bacteria; (C) Oke nje bacteria n'ime oke nje bacteria n'ime oke nje bacteria. A na-egosi data dị ka nkezi ± SEM (n = 6).#p < 0.05 ma ọ bụ ###p < 0.001 vs otu njikwa (Con); *p < 0.05 ma ọ bụ **p < 0.01 ma ọ bụ ***p < 0.001 vs otu DSS.
Ebe ọ bụ na obere abụba dị mkpụmkpụ (SCFAs) bụ ihe ndị bụ isi na-emepụta ihe dị na Akkermansia na Prevotellaceae_UCG-001, ebe acetate, propionate na butyrate bụ SCFA kachasị na lumen eriri afọ, anyị ka nọ na ọmụmụ ihe anyị. Dịka egosiri na Foto 5, e belatara oke fecal acetate, propionate, na butyrate n'ime otu DSS gwọrọ, ebe ọgwụgwọ BLG nwere ike igbochi mbelata a nke ukwuu.
Foto nke 5. BLG na-eme ka ọkwa SCFA dị na nsị nke òké nwere colitis nke DSS kpatara.(A) Ọdịnaya Acetic acid dị na nsị; (B) Ọdịnaya Propionic acid dị na nsị; (C) Ọdịnaya Butyric acid dị na nsị. E gosipụtara data dị ka nkezi ± SEM (n = 6).#p < 0.05 ma ọ bụ ##p < 0.01 vs otu njikwa (Con); *p < 0.05 ma ọ bụ **p < 0.01 vs otu DSS.
Anyị gbakọọkwara njikọ Pearson n'etiti ọdịiche dị n'etiti SCFA na microbiota fecal. Dịka egosiri na Foto nke 6, Akkermansia nwere njikọ dị mma na mmepụta nke propionic acid (Pearson = 0.4866) na butyric acid (Pearson = 0.6192). N'ụzọ dị iche, ma Enteromonas na Oscillobacter nwere njikọ na-adịghị mma na mmepụta acetate, yana Pearson coefficients nke 0.4709 na 0.5104, n'otu n'otu. N'otu aka ahụ, Ruminokoccaceae_UCG-014 nwere njikọ na-adịghị mma na mmepụta nke propionic acid (Pearson = 0.4508) na butyric acid (Pearson = 0.5842), n'otu n'otu.
Foto nke 6. Nyocha njikọ Pearson n'etiti SCFA dị iche iche na nje bacteria colonic.(A) Enteromonas nwere acetic acid; (B) Concussion bacillus na acetic acid; (C) Akkermansia vs propionic acid; (D) Ruminococcus_UCG-014 na propionic acid; (E) Akkermansia na butyric acid; (F) ) Ruminococcus _UCG-014 na butyric acid.
Peptide-1 dị ka Glucagon (GLP-1) bụ ngwaahịa proglucagon (Gcg) nke a na-akpọ "post-translational cell" nke nwere ihe mgbochi mkpali.28 Dịka egosiri na Foto 7, DSS butere mbelata dị ukwuu na ngosipụta Gcg mRNA. Ọgwụgwọ colon na BLG nwere ike ịgbanwe mbelata Gcg nke DSS kpatara nke ukwuu ma e jiri ya tụnyere otu njikwa (Fig. 7A). N'otu oge ahụ, ọkwa nke GLP-1 na serum belatara nke ukwuu na otu DSS gwọrọ, ọgwụgwọ BLG nwekwara ike igbochi mbelata a nke ukwuu (Fig. 7B). Ebe ọ bụ na obere abụba acids nwere ike ịkpalite mwepụta GLP-1 site na mkpali G-protein-coupled receptor 43 (GRP43) na G-protein-coupled receptor 41 (GRP41), anyị nyochakwara GPR41 na GRP43 na colon nke oke colitis ma chọpụta na ngosipụta mRNA colonic nke GRP43 na GPR41 belatara nke ukwuu mgbe nsogbu DSS gasịrị, ọgwụgwọ BLG nwekwara ike ịzọpụta mbelata ndị a nke ọma (Foto 7C na D).
Foto nke 7 BLG na-eme ka ọkwa GLP-1 dị n'ọbara na ngosipụta mRNA Gcg, GPR41 na GRP43 dị n'ime òké ndị DSS gwọrọ.(A) Ngosipụta mRNA Gcg dị n'ime anụ ahụ eriri afọ; (B) Ọkwa GLP-1 dị n'ime serum; (C) Ngosipụta mRNA GPR41 dị n'ime anụ ahụ eriri afọ; (D) Ngosipụta mRNA GPR43 dị n'ime anụ ahụ eriri afọ. A na-egosi data dị ka nkezi ± SEM (n = 5–6).#p < 0.05 ma ọ bụ ##p < 0.01 dị n'ime otu njikwa (Con); *p < 0.05 dị n'ime otu DSS.
Ebe ọ bụ na ọgwụgwọ BLG nwere ike ime ka ọkwa GLP-1 dị n'ọbara, ngosipụta mRNA nke colonic Gcg, na ọkwa SCFA nke fecal dị n'ime òké ndị DSS gwọrọ, anyị nyochakwara acetate, propionate, na butyrate yana site na njikwa (F-Con), DSS colitis (F-Con) -DSS) na òké ndị BLG gwọrọ colitis (F-BLG) mgbe ewepụtara GLP-1 site na mkpụrụ ndụ epithelial murine colonic mbụ. Dịka egosiri na Foto 8A, mkpụrụ ndụ epithelial nke oke bekee mbụ ejiri 2 mM acetic acid, propionic acid, na butyric acid gwọọ, n'otu n'otu, kpaliri mwepụta GLP-1 nke ukwuu, dịka ọmụmụ ihe ndị gara aga si dị.29,30 N'otu aka ahụ, F-Con, F-DSS, na F-BLG niile (nke ruru 0.25 g nke nsị) kpaliri mwepụta nke GLP-1 nke ukwuu site na mkpụrụ ndụ epithelial murine colonic mbụ. Nke kachasị mkpa bụ na ọnụọgụ GLP-1 nke mkpụrụ ndụ epithelial nke oke bekee mbụ gwọrọ F-DSS dị obere karịa nke F-Con na F-BLG gwọrọ mkpụrụ ndụ epithelial nke oke bekee. (Foto 8B). Data ndị a na-egosi na ọgwụgwọ BLG weghachiri mmepụta GLP-1 sitere na SCFA n'ime eriri afọ nke ukwuu.
Foto nke 8 SCFA sitere na BLG na-akpali ntọhapụ GLP-1 site na mkpụrụ ndụ epithelial murine nke mbụ.(A) Acetic acid, propionic acid, na butyric acid kpaliri ntọhapụ nke GLP-1 site na mkpụrụ ndụ epithelial murine nke mbụ; (B) ihe ndị sitere na nsị F-Con, F-DSS na F-BLG nke mkpụrụ ndụ epithelial murine nke mbụ kpaliri Ọnụọgụ nke GLP-1 ewepụtara. E tinyere mkpụrụ ndụ epithelial colonic n'ime efere petri iko-ala ma jiri 2 mM acetic acid, propionic acid, butyric acid, na ihe ndị sitere na nsị F-Con, F-DSS, na F-BLG (nke hà nhata 0.25 g nsị), n'otu n'otu. Awa abụọ na 37°C, 5% CO2, n'otu n'otu. ELISA chọpụtara ọnụọgụ GLP-1 ewepụtara na mkpụrụ ndụ epithelial murine nke bụ isi. A na-egosi data dị ka nkezi ± SEM (n = 3).#p < 0.05 ma ọ bụ ##p < 0.01 vs. blank ma ọ bụ F-Con; *p < 0.05 vs. F-DSS.
Mkpirisi: Ace, acetic acid; Pro, propionic acid; agbanyeghị, butyric acid; F-Con, ihe e si na nsị wepụta site na òké njikwa; F-DSS, ihe e si na nsị wepụta site na òké colitis; F-BLG, site na eriri afọ ejiri BLG gwọọ ihe e si na nsị wepụta site na òké na-akpata mbufụt.
N'ịbụ nke Òtù Ahụ Ike Ụwa depụtara dị ka ọrịa na-anaghị emegharị ahụ, UC na-aghọ ihe ize ndụ zuru ụwa ọnụ; Agbanyeghị, ụzọ dị irè maka ịkọ amụma, igbochi, na ịgwọ ọrịa ahụ ka dị oke. Ya mere, ọ dị mkpa ịchọpụta ma mepụta atụmatụ ọgwụgwọ ọhụrụ dị nchebe ma dị irè maka UC. Nkwadebe ọgwụ ọdịnala nke ndị China bụ nhọrọ na-ekwe nkwa n'ihi na ọtụtụ nkwadebe ọgwụ ọdịnala nke ndị China egosila na ha dị irè n'ịgwọ UC n'ime ndị China kemgbe ọtụtụ narị afọ, ha niile bụ ihe ndị dị ndụ na ihe ndị sitere n'okike nke na-adịghị emerụ ụmụ mmadụ na anụmanụ ahụ ahụ.31,32 Ọmụmụ ihe a chọrọ ịchọ nkwadebe ọgwụ ọdịnala China dị mma ma dị irè maka ọgwụgwọ UC na inyocha usoro ọrụ ya. BLG bụ usoro ọgwụ ọdịnala China a ma ama nke ejiri agwọ influenza.8,33 Ọrụ na ụlọ nyocha anyị na ndị ọzọ egosila na indigo, ngwaahịa ọgwụ ọdịnala China nke e ji otu ihe dị ka BLG mee, na-egosipụta irè dị ukwuu n'ịgwọ UC n'ime mmadụ na anụmanụ.4,34 Agbanyeghị, mmetụta mgbochi colitis nke BLG na mmetụta ya Usoro ahụ edoghị anya. Na ọmụmụ ihe a ugbu a, nsonaazụ anyị na-egosi na BLG na-ebelata mbufụt nke DSS kpatara, nke jikọtara ya na mgbanwe nke microbiota eriri afọ na mweghachi nke eriri afọ sitere na Mmepụta GLP-1.
A maara nke ọma na a na-amata UC site na oge nlọghachi azụ nwere njirimara ahụike nkịtị, dịka mbelata ibu, afọ ọsịsa, ọbara ọgbụgba n'ime eriri afọ, na nnukwu mmebi nke mucosa eriri afọ.35 Ya mere, enyere colitis nlọghachi azụ na-adịghị ala ala site n'inye usoro atọ nke 1.8% DSS maka ụbọchị ise, wee soro ya ụbọchị asaa nke mmiri ọṅụṅụ. Dịka egosiri na Foto 1B, mbelata ibu na-agbanwe agbanwe na akara DAI gosiri mmụba nke colitis nlọghachi azụ na-adịghị ala ala. Òké n'ime otu ejiri BLG gwọọ gosiri mgbake dị elu site na ụbọchị 8, nke dị nnọọ iche na ụbọchị 24. A hụkwara otu mgbanwe ndị ahụ na akara DAI, na-egosi mmụba na mmezi ahụike nke colitis. N'ihe gbasara mmerụ ahụ eriri afọ na ọnọdụ mbufụt, ogologo eriri afọ, mmebi anụ ahụ eriri afọ, na ngosipụta mkpụrụ ndụ ihe nketa na mmepụta nke cytokines proinflammatory TNF-α, IL-1β, na IL-6 na anụ ahụ eriri afọ ka emechara nke ọma mgbe ọgwụgwọ BLG gasịrị. N'ozuzu, nsonaazụ ndị a na-egosi nke ọma na BLG dị irè n'ịgwọ colitis nlọghachi azụ na-adịghị ala ala na òké.
Kedu ka BLG si emetụta mmetụta ọgwụ ya? Ọtụtụ nnyocha gara aga egosila na microbiota eriri afọ na-arụ ọrụ dị mkpa na pathogenesis nke UC, ọgwụgwọ dabere na microbiome na microbiome apụtala dị ka atụmatụ mara mma maka ọgwụgwọ UC. N'ọmụmụ ihe a, anyị gosiri na ọgwụgwọ BLG butere mgbanwe dị ukwuu na nhazi microbiota eriri afọ, na-egosi na mmetụta nchebe nke BLG megide colitis nke DSS kpatara metụtara mgbanwe nke microbiota eriri afọ. Nleba anya a kwekọrọ n'echiche na ịmegharị nhazi homeostasis nke eriri afọ microbiota bụ ụzọ dị mkpa iji ghọta irè nke nkwadebe TCM.36,37 N'ụzọ dị mkpa, Akkermansia bụ nje bacteria Gram-negative na nke anaerobic kpọmkwem nke bi na oyi akwa mucous nke eriri afọ, nke na-emebi mucins, na-emepụta propionic acid, na-akpali nhazi nke mkpụrụ ndụ goblet, ma na-edobe mucosa. ọrụ nke iguzosi ike n'ezi ihe mgbochi.26 Ọtụtụ data ahụike na anụmanụ na-atụ aro na Akkermansia nwere njikọ dị ukwuu na mucosa dị mma,38 na nchịkwa ọnụ nke Akkermansia spp. nwere ike ime ka mbufụt mucosal ka mma nke ukwuu.39 Data anyị ugbu a na-egosi na ọnụọgụ Akkermansia dị ukwuu na-abawanye nke ukwuu mgbe a gwọchara BLG. Na mgbakwunye, Prevotellaceae_UCG-001 bụ nje bacteria na-emepụta SCFA.27 Ọtụtụ ọmụmụ gosiri na a hụrụ Prevotellaceae_UCG-001 n'ọtụtụ dị ala na nsị anụmanụ nwere colitis.40,41 Data anyị ugbu a na-egosikwa na ọgwụgwọ BLG nwere ike ime ka mmụba nke Prevotellaceae_UCG-001 dị ukwuu na eriri afọ nke òké ndị DSS gwọrọ. N'ụzọ dị iche, Oscillibacter bụ nje bacteria mesophilic, nke na-anaghị anabata onwe ya nke ọma.42 kọrọ na ọnụọgụ Oscillibacter dị ukwuu na òké UC ma nwee njikọ dị mma na ọkwa IL-6 na IL-1β na akara ọrịa.43,44 N'ụzọ dị mkpa, ọgwụgwọ BLG belatara nke ukwuu ọnụọgụ Oscillibacter dị na nsị nke òké ndị DSS gwọrọ. N'ụzọ dị mkpa, nje bacteria ndị a gbanwere na BLG bụ nje bacteria kachasị emepụta SCFA. Ọtụtụ ọmụmụ ihe gara aga egosila mmetụta bara uru nke SCFAs nwere ike inwe. gbasara mbufụt eriri afọ na nchekwa nke epithelial nke eriri afọ.45,46 Ihe ndekọ anyị ugbu a hụkwara na oke nke SCFA acetate, propionate, na butyrate na nsị DSS gwọrọ mụbara nke ukwuu na òké BLG gwọrọ. Mgbe a gbakọtara ha ọnụ, nchọpụta ndị a na-egosi nke ọma na ọgwụgwọ BLG nwere ike ime ka nje bacteria na-emepụta SCFA nke DSS gwọrọ dịkwuo mma n'ime òké ndị nwere colitis na-alaghachi azụ ogologo oge.
GLP-1 bụ incretin a na-emepụtakarị na ileum na colon ma na-arụ ọrụ dị mkpa n'igbu oge mmịpụta afọ na ibelata glucose ọbara mgbe emechara nri.47 Ihe akaebe na-egosi na dipeptidyl peptidase (DPP)-4, onye na-anabata GLP-1, na ọgwụ nanomedicine GLP-1 nwere ike ibelata mbufụt eriri afọ na òké nke ọma.48-51 Dịka akọpụtara na ọmụmụ ihe ndị gara aga, enwere njikọ dị elu na ọkwa GLP-1 dị na plasma na ụmụ mmadụ na òké. 52 Data anyị ugbu a na-egosi na mgbe ọgwụgwọ BLG gasịrị, ọkwa GLP-1 dị na serum na ngosipụta Gcg mRNA mụbara nke ukwuu. N'otu aka ahụ, mmepụta GLP-1 mụbara nke ukwuu na omenala colonic mgbe mkpali na ihe ndị sitere na òké colitis ejiri BLG gwọọ ma e jiri ya tụnyere mkpali na ihe ndị sitere na òké colitis ejiri DSS gwọọ. Kedu ka SCFA si emetụta ntọhapụ nke GLP-1?Gwen Tolhurst et al. kọrọ na SCFA nwere ike ịkpalite mmepụta GLP-1 site na GRP43 na GPR41.29 Data anyị dị ugbu a na-egosikwa na ọgwụgwọ BLG na-eme ka mmịpụta mRNA nke GRP43 na GPR41 dịkwuo elu nke ukwuu n'ime eriri afọ nke òké ndị DSS gwọrọ. Data ndị a na-egosi na ọgwụgwọ BLG nwere ike iweghachi mmepụta GLP-1 nke SCFA kwalitere site na ịgbalite GRP43 na GPR41.
BLG bụ ọgwụ a na-ere n'ọnụ ahịa ogologo oge (OTC) na China. Oke kachasị anabatara nke BLG n'ime òké Kunming bụ 80g/Kg, a hụbeghịkwa nnukwu nsí.53 Ugbu a, oke akwadoro nke BLG (na-enweghị shuga) n'ime mmadụ bụ 9-15 g/ụbọchị (ugboro atọ n'ụbọchị). Nnyocha anyị gosiri na BLG na 1g/Kg nke DSS kpatara na-ebelata ọrịa colitis na-adịghị ala ala. Oke a dị nso na oke BLG ejiri mee ihe n'ụlọọgwụ. Ọmụmụ ihe anyị chọpụtakwara na usoro ọrụ ya na-agbanwe, ma ọ dịkarịa ala n'akụkụ ụfọdụ, site na mgbanwe na microbiota eriri afọ, karịsịa nje bacteria na-emepụta SCFA, dị ka Akkermansia na Prevotellaceae_UCG-001, iji weghachi mmepụta GLP-1 sitere na eriri afọ. Nchọpụta ndị a na-egosi na BLG kwesịrị ịtụlekwu dị ka ihe nwere ike ịgwọ ọrịa colitis. Agbanyeghị, usoro kpọmkwem nke o ji emezi microbiota eriri afọ ka ga-akwado site na òké na-enweghị microbiota na ntinye nje bacteria na nsị.
Ace, acetic acid; but, butyric acid; BLG, pandan; DSS, dextran sodium sulfate; DAI, index ọrụ ọrịa; DPP, dipeptidyl peptidase; FID, ihe nchọpụta ionization ọkụ; F-Con, njikwa ihe ndị sitere na fecal nke òké; F-DSS, ihe ndị sitere na fecal nke òké DSS colitis; F-BLG, ihe ndị sitere na fecal nke òké colitis a gwọchara 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 Salt Solution; OTC, OTC; PCA, isi ihe nyocha; PCoA, isi nyocha nhazi; Pro, propionic acid; SASP, sulfasalazine; SCFA, obere abụba acids; ọgwụ China, ọgwụ China ọdịnala; UC, ulcerative colitis.
Kọmitii Ụkpụrụ Omume Anụmanụ nke Mahadum Peking Shenzhen-Hong Kong nke Mahadum Sayensị na Teknụzụ (Shenzhen, China) kwadoro usoro nnwale niile dịka Ntuziaka Ụlọ Ọrụ na Iwu Anụmanụ si dị (nọmba omume A2020157).
Ndị edemede niile tinyere aka dị ukwuu n'ichepụta na imepụta ihe, inweta data, ma ọ bụ nyocha data na nkọwa; sonyere na ide edemede ahụ ma ọ bụ imegharị ihe dị mkpa n'ime ọgụgụ isi; kwenyere izipu akwụkwọ edemede ahụ na akwụkwọ akụkọ ugbu a; mechara kwado mbipụta ahụ maka mbipụta; Ọ bụ ọrụ maka akụkụ niile nke ọrụ.
Ọrụ a bụ nke National Natural Science Foundation nke China (81560676 na 81660479), ọrụ klas mbụ nke Mahadum Shenzhen (86000000210), Shenzhen Science and Technology Innovation Committee Fund (JCYJ20210324093810026), na Guangdong Provincial Medical Science and Technology Research Fund (A2020157 na A2020272), Guizhou Medical University Pharmacy Guizhou Province nke Key Laboratory (YWZJ2020-01) na Peking University Shenzhen Hospital (JCYJ2018009) kwadoro.
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