| Objectives To explore gut microbiota dysregulation affecting in secondary bile acid metabolism,Th17/Treg immune balance,autoimmune levels,SLE disease activity,and efficacy mechanism of Jiedu Quyu Ziyin Prescription.Methods 1.Animal experiments:42 specific pathogen free(SPF)female MRL/Ipr spontaneous lupus mice were divided into the model,Glucocorticoid,traditional chinese medicine,traditional chinese medicine with Glucocorticoid,3-oxoLCA,isoalloLCA,and 3-oxoLCA+isoalloLCA groups(6 mice/group).Another 6 MRL/MpJ mice were selected as the control group.All drugs were administered once a day by oral gavage from the 7th week to the 14th week.The spleen and lymph node indices were calculated.The kidneys were removed for renal pathology slides,such as HE staining,immunohistochemistry(IHC),and immunofluorence(IF)staining.The serum specimens were processed for ELISA examination to detect the levels of Anti ds-DNA,ANA,IL-17A,and IL-6.Flow cytometry expression analysis of Th17 and Treg cells from spleen,and calculating Th17/Treg ratio.RNA was prepared from those cells and the expression levels of ROR-yt and Foxp3 mRNA were examined by RT-qPCR,and the expression of 3α/β-HSD mRNA from the colon was detected as well.16s RNA gene sequencing detected gut microbiota community change at the genus level.Multi-dimensional mass spectrometry-based shotgun lipidomics(MDMS-SL)technology was used to test the levels of 3-oxoLCA and IsoalloLCA from feces.2.In vitro experiment of Secondary bile acid 3-oxoLCA and isoalloLCA regulating Th17 and Treg differentiation:Naive CD4+T cells were sorted from the spleen of MRL/lpr mice,stimulated,and differentiated into Th17 or Treg cells.Divided into Glucocorticoid,traditional Chinese medicine,3-oxoLCA,and isoalloLCA group.After treatment with corresponding drugs for 96 hours,Flow cytometry tested the Th17/Treg ratio,the expression level of ROR-yt and Foxp3 protein was detected by Western-Blot,the expression levels of ROR-yt and Foxp3 mRNA were examined by RT-qPCR.3.Microbiota transplants experiment of gut microbiota regulate 3-oxoLCA and isoalloLCA metabolic:12 MRL/lpr mice were randomly divided into the model group and the SLE fecal microbiota transplantation(SLEFMT)group,another 6 MRL/MpJ mice were selected as the control group.Since the 4th week,the SLEFMT group was treated with antibiotics every other day 3 times to deplete the gut microbiota.From the 5th week,the fecal microbiota transplantation was performed every other day 5 times.The spleen and lymph node indices were calculated,and the kidneys were removed for renal pathology slides,such as HE staining,immunohistochemistry(IHC),and immunofluorence(IF)staining.The serum specimens were processed for ELISA examination to detect the levels of Anti ds-DNA,IL-10,and TNF-α.The expression of 3α/β-HSD mRNA from the colon was detected by RT-qPCR,and 16s RNA gene sequencing detected gut microbiota community change at the genus level.MDMS-SL technology was used to test the levels of 3-oxoLCA and isoalloLCA in the stool.Results 1.Results from animal experiments:compared with the model group,the swollen lymph node and enlarged spleen were released after treatment,and renal pathologies such as C3 and IgG deposition were mitigated as well.The levels of autoantibody and inflammation significantly decreased.The flow cytometry result suggested that the proportion of Th17s and Tregs,and the Th17/Treg ratio in the spleen were decreased after treatment.RT-qPCR result showed that ROR-yt mRNA expression level declined in the treatment groups,and Foxp3 mRNA expression level increased.16s RNA gene sequencing result revealed that there was a significant difference in the beta diversity of gut microbiotas in the model group,and the structure of gut microbiotas showed a difference between the model group and the treatment groups,while 3α-HSD mRNA expression levels were elevated and 3β-HSD exhibited no significant change.Besides,the lipidomic analysis showed that Jiedu Quyu Ziyin Prescription(JP)intervention resulted in an increase of 3-oxoLCA and isoalloLCA.2.Results from in vitro experiments:The flow cytometry result suggested that 3-oxoLCA inhibited Th17 cell differentiation,and isoalloLCA promoted Treg cell differentiation.Western blot confirmed that ROR-γt protein expression decreased in the 3-oxoLCA group,and Foxp3 protein expression increased in the isoalloLCA group.RT-qPCR result showed that 3-oxoLCA suppressed ROR-γt mRNA expression,and isoalloLCA promoted Foxp3 mRNA expression.In addition,the JP group showed no significant change in Th17 and Treg differentiation.3.Results from fecal microbiota transplantation experiments:Compared to the model group,the spleens and lymph nodes of mice were enlarged after the transplantation of feces from SLE patients.Renal pathology was aggravated.The levels of autoantibody and inflammation significantly increased.16s RNA gene sequencing result revealed that there was a significant difference in the beta diversity of gut microbiotas between the groups,and the structure of gut microbiotas showed a difference.RT-qPCR result showed that 3α/β-HSD mRNA expression levels decreased in the SLEFMT group.Lipidomic analysis showed that there was a downward trend in the 3-oxoLCA content.Conclusion 1.The disturbance of gut microbiota resulted in secondary bile acid 3-oxoLCA and isoalloLCA metabolic disorder,Th17/Treg immune balance dysregulation,which has an important role in the development and progression of SLE.2.Jiedu Quyu Ziyin Prescription could effectively modulate the structure and function of the gut microbiota,raise the expression of 3α/β-HSD,restore secondary bile acid 3-oxoLCA and isoalloLCA metabolic,regulate the imbalance of Th17/Treg immune,reduce inflammation response and disease activity,thus alleviate the condition. |
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