The Mechanism Of Action Of Polysaccharides From Polygonatum Sibiricumin The Treatment Of COPD By Regulating Th17/Treg Balance Based On The Gut-Lung Axis

The theory of“The lung and the large intestine being interior-exteriorly related“in Chinese medicine and modern studies on the“gut–lung“axis have confirmed the close relationship between the intestine and the lung,with the balance of Th17/Treg cells being the focus of the association.The PSP-NP is a neutral polysaccharide isolated and purified from Polygonatum sibiricumin.Previous studies have shown that PSP-NP can protect intestinal mucosal barrier,inhibit inflammation and regulate Th17/Treg-related cytokines.Therefore,it is speculated that PSP-NP may regulate the balance of Th17/Treg cells to exert the effect of co-treatment of intestine and lung.In this study,a mouse model of chronic obstructive pulmonary disease was constructed to examine the preventive and curative effects and mechanisms of PSP-NP on the intestine and lung by observing their lung and intestine pathological histology,mucosal barrier,inflammatory factor content related to Th17/Treg balance,the number and ratio of Th17/Treg cells,diversity of gut microbiota and content of short-chain fatty acid.We aim to demonstrate whether it exerts intestinal and pulmonary cotreatment by regulating the Th17/Treg balance,to elucidate its pathway to regulate Th17/Treg balance,and to further investigate the mechanism of simultaneous treatment of lung and intestine of PSP-NP through Th17/Treg balance.The study methods and results are as follows:1.Effect of PSP-NP on growth performance and clinical manifestations of COPD miceSeventy-five C57BL/6j mice were randomly divided into 5 groups:Control,Model,PSP-NP-low,middle and high dose group（50/100/200 mg/kg/d）.The COPD model was established by exposuring to cigarette smoke combined with LPS nasal drops for experimental mice except for the control group.LPS（2.5 mg/m L）was administered on day1 and day 14 of the experiment,respectively,and the rest of the time exposure to cigarette smoke normally（30 min/10 sticks/time,6 days/week）.During the construction of the model,PSP-NP was administered by gavage according to the group once a day in the morning with a gavage volume of 0.1m L/10g,and the control and model groups were instilled with the same volume of saline for a test period of 10 weeks.During the experiment,the mice were weighed once a week,while behavioral changes and clinical signs were recorded.The results showed that COPD mice were dyspnoeic,depressed,slow to move and slow to gain weight,after PSP-NP administration,clinical symptoms recovered and weight increased.2.The effect of PSP-NP on the lungs of COPD miceSamples were collected at the end of the experiment to observe the effect of PSP-NP on the lung tissue of COPD model mice.（1）Effect on lung function:PH,PCO₂ and HCO₃in serum were analysed by blood gas analyser.（2）Effect on histopathological changes in lung tissue:HE staining was used to observe the histopathological changes in lung tissue,and Image J software was used to determine the number and diameter of alveoli and calculate the ratio of number and diameter of alveoli.（3）Effect on mucosal barrier:The contents of ZO-1 and Occludin were measured by enzyme-linked immunosorbent assay（ELISA）in lung tissue;（4）Effect on inflammatory cytokines:The content of inflammatory factors interleukin6,17,21,23,10 and tumour necrosis factorα（TNF-α）were determined by ELISA.（5）Effect on anti-inflammatory cytokines:The content of anti-inflammatory factors IL-2,IL-10 and transforming growth factorβ（TGF-β）were determined by ELISA.The results showed that:（1）the results of blood gas analysis showed that the levels of PCO₂,HCO₃ in the COPD model mice increased significantly（P<0.05）,the level of PCO2,HCO₃decreased significantly（P<0.05）after the administration of PSP-NP,and the lung function was improved.（2）In COPD model mice,the number of alveoli was significantly reduced,the alveolar septum was severely thickened,some of the alveolar septum was broken,inflammatory cells were infiltrated,diffuse hemorrhage was observed,and the alveolar number to diameter ratio was significantly increased（P<0.05）.All of the above symptoms were significantly improved and the alveolar number to diameter ratio was significantly reduced（P<0.05）after administration of PSP-NP.（3）The content of tight junction protein ZO-1 was highly significantly reduced（P<0.001）and Occludin was significantly reduced（P<0.05）in the COPD model group mice,and the content of both ZO-1 and Occludin recovered after PSP-NP adminis0tration,with Occludin being highly significantly increased in the high-dose group（P<0.01）.（4）The contents of pro-inflammatory factors IL-6,IL-17,IL-21,IL-23 and TNF-αwere highly significantly increased（P<0.01）in COPD model mice,and the contents of pro-inflammatory factors were significantly decreased（P<0.05）after PSP-NP administration in a dose dependent manner.（5）The content of anti-inflammatory factors IL-2 and TGF-βwere significantly reduced（P<0.05）,IL-10 was increased（P>0.05）but not significantly in COPD mice,the content of IL-2,IL-10 and TGF-βwere significantly increased（P<0.05）after PSP-NP administration.3.The effect of PSP-NP on the intestinal tract of COPD miceSamples were collected to observe the effect of PSP-NP on the intestine.（1）Effect on histopathology of jejunum:HE staining was used to observe the pathological changes of jejunum.（2）Effect on histopathology of colon:HE staining was used to observe the pathological changes of colon.（3）The effect on mucosal barrier and permeability:the goblet cells of jejunum were counted by AB-PAS staining;ELISA was used to determine the contents of the tight junction protein ZO-1,Occludin in intestinal tissue and lipopolysaccharide（LPS）in serum.（4）Effect on pro-inflammatory cytokines:The contents of inflammatory cytokines IL-6,IL-17,IL-21,IL-23 and TNF-αwere measured by ELISA.（5）Effects on anti-inflammatory cytokines:The contents of anti-inflammatory factors IL-2,IL-10 and TGF-βwere determined by ELISA.The results showed that:（1）Effects on jejunal histopathology:COPD model mice had dysplastic jejunal villi and detached intestinal villi epithelium,with a highly significant increase in histological score（P<0.001）;after PSP-NP administration,the histological score was significantly reduced（P<0.05）and the pathological changes were significantly improved.（2）Effects on colonic histopathology:the number of colonic glands was significantly reduced in the COPD model group mice,accompanied by inflammatory cell infiltration,and the histological score was highly significantly increased（P<0.01）,after PSP-NP administration,the histological score was significantly reduced（P<0.05）,and the colonic pathological changes were significantly improved.（3）Effects on mucosal barrier and permeability:jejunal goblet cells were significantly reduced in the COPD model group（P<0.01）;the contents of jejunal and colonic tight junction proteins ZO-1 and Occludin were significantly reduced（P<0.001）;the serum LPS content was significantly increased（P<0.001）.After PSP-NP administration,the number of jejunal cupped cells increased significantlyi in the medium and high dose groups（P<0.001）;the content of jejunal and colonic tight junction protein increased significantly（P<0.05）;and the serum LPS content decreased significantly（P<0.05）in the medium and high dose groups.（4）Effects on pro-inflammatory cytokines:the contents of pro-inflammatory factors IL-6,IL-17,IL-21,IL-23and TNF-αwere all significantly increased in COPD model mice（P<0.001）,and the contents of pro-inflammatory factors were all significantly decreased after PSP-NP administration（P<0.05）;（5）the effect on anti-inflammatory cytokines:the content of IL-2was not significantly decreased（P>0.05）,the content of Il-10 was not significantly increased（P>0.05）,the content of TGF-βwas significantly decreased（P<0.05）;the contents of anti-inflammatory factors were all significantly increasedafter PSP-NP administration（P<0.05）.4.The effect of PSP-NP on the balance of Th17/Treg cell in COPD miceThe expression of Th17 signature protein RAR-related orphan receptorγT（ROR-γt）and Treg signature protein fork head box protein P3（Foxp3）in lung and colon tissues were measured by Western Blot（WB）and immunohistochemistry;the number of Th17/Treg cells in serum was detected by flow cytometry and the ratio of the Th17/Treg was calculated.Both WB and immunohistochemical results showed that in COPD mice,lung and colon ROR-γt expression was significantly increased（P<0.05）,Foxp3 expression was significantly decreased（P<0.05）,ROR-γt to Foxp3 ratio was highly significantly increased（P<0.01）,and after PSP-NP administration,both lung and colon ROR-γt expression was significantly decreased（P<0.05）,Foxp3 expression was significantly increased（P<0.05）,and the ROR-γt to Foxp3 ratio was highly significantly decreased（P<0.001）,with consistent changes in lung and colon tissues.The results of serum flow assay showed that the number of Th17 cells was highly significant increased in COPD mice（P<0.001）,the number of Treg was decreased but not significant（P>0.05）,the ratio of the Th17/Treg was highly significant increased（P<0.001）;the number of Th17 cells was highly significant decreased after PSP-NP administration（P<0.001）,the number of Treg was highly significant increased in the low and high dose groups（P<0.01）,and the ratio of the two was significantly decreased（P<0.001）.The trend was consistent with that of lung and colon,and the trend was more significant in serum.5.Effect of PSP-NP on diversity of gut microbiota and short-chain fatty acids in COPD miceThe changes in the diversity of colonic contents of COPD mice were measured by16Sr RNA high-throughput sequencing;the content of short-chain fatty acids（SCFAs）was determined by gas chromatography;Spearman correlation coefficients were used to analyze the correlations between gut microbiota and SCFAs,gut microbiota and Th17/Treg,SCFAs and Th17/Treg.The results showed that:（1）the effect on gut microbiota diversity:the abundance of gut microbiota increased and the diversity decreased in mice of COPD model group,and the abundance and diversity of gut microbiota increased significantly after PSP-NP administration.（2）Effects on gut microbiota composition and significant species:After PSP-NP administration,the abundance of Firmicutes decreased and the abundance of Bacteroidetes increased,and the ratio of the two was highly significant（P<0.001）;the abundance of Akkermansia and Dubosiella increased.The significant species in the PSP-NP administration group were Bacteroidetes and Dubosiella.Among them,Firmicutes were positively correlated with the number of Th17 cells and Th17/Treg ratio and negatively correlated with the number of Treg in serum,lung and colon tissues of COPD mice;Bacteroidetes,Akkermansia and Dubosiella were negatively correlated with the number of Th17 cells and Th17/Treg ratio and positively correlated with the number of Treg in serum,lung and colon tissues of COPD mice.（3）Effects on the content of SCFAs:The content of short-chain fatty acids in the model group,except for butyric acid and valeric acid,was significantly reduced（P<0.01）.Acetic acid,propionic acid,isobutyric acid and isovaleric acid were significantly increased after PSP-NP administration（P<0.5）;They are all were positively correlated with Akkermansia and Dubosiella were positively correlated;among them,the levels of acetic acid,propionic acid and isobutyric acid were negatively correlated with the number of Th17 cells and Th17/Treg ratio,positively correlated with the number of Treg in serum,lung and colon tissues of COPD mice.6.Study on the therapeutic effect of FMT on COPD miceThirty mice were divided into three groups of 10 mice each,the model group（C）,COPD model group（M）and Fecal microbiota transplantation group（FMT）.The COPD model was established by exposuring to cigarette smoke combined with LPS nasal drops in group M and FMT.LPS（2.5 mg/m L）was administered on day 1 and day 14 of the experiment,respectively,and the rest of the time exposure to cigarette smoke normally（30min/10 sticks/time,6 days/week）.While constructing the model,the FMT group administered fecal supernatant by gavage once a day in the morning with a gavage volume of 0.1m L/10g,the C and M groups administered the same volume of saline for a 10-week trial period.（1）Effects on pathological changes:HE staining was used to observe the histopathological changes in lung,jejunum and colon.（2）Effects on mucosal barrier and permeability:ELISA kits were used to determine the contents of lung,jejunum and colon tight junction protein ZO-1,Occludin and serum LPS;（4）Effects on pro-inflammatory cytokines:ELISA was used to determine the contents of inflammatory cytokines IL-6,IL-17,IL-21,IL-23 and TNF-α.（5）Effects on anti-inflammatory cytokines:The levels of anti-inflammatory factors IL-2,IL-10 and TGF-βwere determined by ELISA.The results showed that:（1）the effects on histopathological changes:the number of alveoli was significantly reduced and the alveolar septum was severely thickened in the lungs of COPD model mice,the ratio of alveolar diameter to number was highly significant higher（P<0.001）;the jejunal villi were severely stunted and the intestinal villi were detached,the number of glands in the colon was reduced and inflammatory cells were infiltrated both jejunal and colonic tissue scores were highly significant higher（P<0.01）.The pathological changes in the lungs,jejunum and colon were significantly improved after FMT;（2）Effects on mucosal barrier and permeability:the contents of tight junction protein ZO-1 and Occludin in lung,jejunum and colon tissues were significantly increased（P<0.05）and LPS was highly significantly decreased（P<0.01）after FMT.（3）Effects on pro-inflammatory cytokines:the content of pro-inflammatory factors IL-6,IL-17,IL-21,IL-23,and TNF-αwere highly significantly increased in COPD mice group（P<0.01）,and the content of pro-inflammatory factors were highly significantly decreased after FMT（P<0.01）.（3）Effects on pro-inflammatory cytokines:the contents of pro-inflammatory factors IL-6,IL-17,IL-21,IL-23 and TNF-αwere highly significantly increased in COPD mice（P<0.01）and highly significantly decreased after FMT（P<0.01）;（4）Effects on anti-inflammatory cytokines:the content of anti-inflammatory factors IL-2 and TGF-βwere significantly reduced in COPD mice（P<0.05）,and the content of IL-10 was not significantly increased（P>0.05）;after FMT,the content of anti-inflammatory factors were all significantly increased（P<0.05）.The results were consistent with the effect of the high dose group of PSP-NP.In summary,PSP-NP can improve growth performance and clinical symptoms in COPD model mice,improve lung and intestinal function by affecting the pathological state of lung and intestinal tissues,enhancing the mucosal barrier,reducing pro-inflammatory factors,and increasing anti-inflammatory factors.PSP-NP also significantly reduced the Th17/Treg cell ratio in lung,intestinal tissues and serum,its regulatory mechanism may be related to improving intestinal diversity of gut microbiota,increasing short-chain fatty acid content and regulating Th17/Treg cell-related cytokines.This experiment provides a basis for the elucidation of the mechanism of intestinal and pulmonary cotreatment of PSP-NP,and it supports the modern theory of"gut-lung"axis and the TCM theory that"the lung and the large intestine being interior-exteriorly related".