| 摘要: | 背景：氣喘是一種支氣管慢性發炎的呼吸道疾病，可能受到腸道微生物群失調的影響，這會破壞免疫平衡和增加肺部發炎的反應。雖然在急性發炎反應期，Inter-alpha-trypsin inhibitor chain 4 (ITIH4) 的表現量會上升，但其在氣喘中所扮演的角色仍不明確。我們的研究主旨在探討ITIH4在氣喘治療中的潛力以及對肺腸軸中微生物群的影響。
材料與方法：我們使用六週大的BALB/c小鼠中建立以ovalbumin (OVA) 誘導氣喘動物模型，隨機將小鼠分成以下5組：Control組、OVA組、2.5 μg/ml ITIH4組、OVA+1.25 μg/ml ITIH4組，以及OVA+ 2.50 μg/ml ITIH4組。OVA組、OVA+1.25 μg/ml ITIH4組，以及OVA+2.50 μg/ml ITIH4組以腹腔注射的方式給予OVA，Control組和2.5 μg/ml ITIH4組則給予等量PBS。每次腹腔注射後間隔一週，總共施打4次。第一周濃度為50 μg/kg OVA，第二周到第四周濃度為25 μg/kg OVA，施打體積均為200 μL。接著，連續七天以鼻腔給藥的方式給予不同劑量的ITIH4分別為1.25 μg/ml和2.5 μg/ml ITIH4。給藥後，連續三天以鼻腔給予100 μg/kg OVA，每次200 μL。第32天時測量血氧濃度和第33天時測量肺功能。利用酵素結合免疫吸附分析法測定支氣管肺泡灌洗液中的細胞激素。使用蘇木精-伊紅染色和K-means 分群測定氣道壁增生以及肺部受損程度。透過氣相質譜儀測定血清中短鏈脂肪酸的濃度和16s DNA序列測定肺部和糞便中的微生物群。
結果：我們發現，ITIH4可以減緩OVA所造成的氣喘小鼠體重下降，並減少氣道阻力以及降低肺部受損程度（p<0.05）。ITIH4降低了肺部IgE、IL-4、IL-5和IL-13的濃度（p<0.05）。血液學分析結果顯示，ITIH4減少了嗜酸性球、淋巴球和單核球的濃度（p<0.05）。另一方面，ITIH4也顯著降低了腸道中IL-4、IL-5和IL-13的表現量（p<0.05）。此外，我們觀察到肺部微生物群中的變化。厭氧菌中的毛螺菌科比例在OVA組別中較低，而在給予ITIH4後顯著增加（p<0.05）。在腸道微生物群的變化中，β多樣性分析顯示接受ITIH4治療的組別與其他組別間有顯著差異（p<0.05）。在陰細菌中，擬桿菌科、螺旋桿菌科和坦納菌科在不同組別間也顯示出顯著差異（p<0.05）。短鏈脂肪酸分析結果顯示，OVA組別中的3-甲基戊酸和4-甲基戊酸濃度較Control組顯著增加（p<0.05），這表明肺和腸道之間存在遠端器官的關聯性。
結論：我們的研究結果顯示ITIH4能夠減輕OVA誘導的氣喘小鼠的過敏反應。我們觀察到肺部微生物群中毛螺菌科以及腸道微生物群中革蘭氏陰性菌（如擬桿菌科、螺旋桿菌科和坦納菌科）的變化對氣喘症狀有顯著影響。這些結果表明，ITIH4可能成為治療氣喘的一種潛在策略。
Introduction
Asthma, a chronic inflammatory respiratory disease, is increasingly understood through the lung-gut axis, suggesting that gut microbiota dysbiosis can exacerbate lung inflammation by disrupting the immune balance. The role of inter-alpha-trypsin inhibitor chain 4 (ITIH4), which is elevated in acute inflammation, in asthma remains uncertain. This study explores ITIH4's therapeutic potential in asthma and its impact through the lung-gut axis.
Materials and methods
We established an ovalbumin (OVA)-induced asthma model in BALB/c mice, divided into five groups: Control, OVA, ITIH4 2.5 μg/ml, OVA + ITIH4 1.25 μg/ml, and OVA + ITIH4 2.5 μg/ml. The OVA groups received weekly OVA injections (50 μg/kg initially, then 25 μg/kg for three weeks), while the Control and ITIH4 groups received PBS. ITIH4 was administered intranasally for seven days (1.25 μg/ml and 2.5 μg/ml), followed by intranasal OVA (100 μg/kg) for three days. Oxygen saturation and lung function were measured. Cytokines in bronchoalveolar lavage fluid (by ELISA), airway thickness (Hematoxylin and eosin), lung tissue damage (K-means clustering), serum short-chain fatty acids (SCFAs by gas chromatography-mass spectrometry), and lung and fecal microbiomes (by 16s DNA sequencing) were determined.
Results
We found that ITIH4 significantly reduced body weight loss, airway resistance, and lung damage in OVA-induced asthmatic mice (p < 0.05). It decreased lung concentrations of IgE, IL-4, IL-5, and IL-13, and lowered levels of eosinophils, lymphocytes, and monocytes (p < 0.05). ITIH4 also reduced IL-4, IL-5, and IL-13 expression in the intestines (p < 0.05) and altered lung microbiota by increasing anaerobic bacteria, particularly Lachnospiraceae (p < 0.05). Gut microbiota beta diversity analysis showed significant differences (p < 0.05), with notable changes in Bacteroidaceae, Helicobacteraceae, and Tannerellaceae proportions (p < 0.05). Short-chain fatty acid analysis revealed higher concentrations of 3-methylpentanoic and 4-methylpentanoic acids in the OVA group compared to the control group (p < 0.05), linking the lung and gut.
Conclusion
Our results suggest that ITIH4 alleviates allergic responses in OVA-induced asthma mice. Significant changes were observed in the lung microbiota, particularly an increase in Lachnospiraceae, and alterations in gut microbiota, including Bacteroidaceae, Helicobacteraceae, and Tannerellaceae. These findings indicate that ITIH4 may be a promising therapeutic strategy for asthma treatment. |
