| Objective:Tripterygium wilfordii is a kind of drug used in clinical treatment.It has a wide range of anti-inflammatory and immunosuppressive activities.Recent studies have confirmed that it has a strong anti-tumor effect.Triptolide(Triptolide,TPL)is the main active ingredient of Tripterygium wilfordii,and it is also the most noticed monomer.Long-term TPL exposure can cause damage to multiple organs such as liver,kidney,spleen or gastrointestinal tract,but the mechanism of damage is still unclear.The cG ASSTING signal pathway is a new natural immune pathway discovered in 2013.The signal pathway product type 1 interferon IFNβ and its downstream effector molecule CXCL10 can participate in the immune damage of liver and kidney tissues.This study focuses on the mechanism of TPL drug-induced renal injury and the role of cGASSTING signaling pathway in the injury process.Methods:1.Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR)was used to detect the effects of TPN and TPL on the expression of inflammatory factors IL-6 and IL-8;Cell Counting Kit-8(CCK8)was used to detects the toxic effects of TPN and TPL on HKC cells.2.Animal experiment:Use TPL monomer to infuse mice,the drug was injected into the abdominal cavity for 6 days,after 6 days,the kidney tissues of 18 mice were processed accordingly for different subsequent experiments.Kidney tissue was made into paraffin sections,HE staining、Immunohistochemical staining、qRT-PCR and Western Blot observe triptolide induces kidney injury in mice and activates the cGASSTING signaling pathway.3.Cell experiment:Through CCK8 experiment、qRT-PCR、Western Blot、immunofluorescence and ELISA observe the phenomenon that triptolide promotes the activation of cGAS-STING signaling pathway in renal epithelial cells.Observe whether knocking down the STING gene can suppress this phenomenon.4.Preliminary mechanism exploration:Through flow cytometry、Western blot and qRT-PCR detection method explored the mechanism of triptolide activating the cGASSTING signal pathway.Results:1.Both TPN and TPL can inhibit the genes expression of inflammatory factors IL-6 and IL-8,TPL has a stronger inhibitory effect on inflammatory factors,but it has a stronger toxic effect on HKC cells.2.The HE staining of mice kidney tissue shows that TPL cause kidney damage in mice;Immunohistochemical staining showed that the expression of STING protein in mice kidney tissue increased with the raising of TPL treatment concentration;The results of qRT-PCR showed that in the high-dose TPL injection group,the expression of STING and IFNβ mRNA significantly increased in the kidney tissue of mice.Western Blot test results showed that the kidney tissue of TPL high-dose injection group mice,the levels of STING,TBK1,IRF3 and their phosphorylation level was significantly increased,also the expression of IFNβ protein increased;3.Cell CCK8 test results show that high concentrations TPL can obviously inhibit the viability of HKC and HK2 cells;qRT-PCR showed that TPL induced highly expression of STING,cGAS,IFNβ and MX1 in HKC and HK2 cells;Western Blot detection results show that the expression of STING protein and the phosphorylation level of TBK1 and IRF3 protein is significantly increased,also the expression of inflammatory factor IFNβ is increased.Immunofluorescence results showed that TPL promoted the translocation of IRF3 protein to the nucleus;ELISA results showed that TPL promoted the secretion of the inflammatory factor IFNβ.knocking down the STING gene,qRTPCR showed that the expression of STING mRNA was reduced,the inflammatory factor IFNβ mRNA was also decreased;Western Blot test results showed that the STING protein and the phosphorylation level of TBK1 and IRF3 protein was significantly reduced,the expression of IFNβ was also reduced.4.Flow cytometry showed that the reactive oxygen species increased in HKC cells;Vitro and vivo experiments,Western Blot detection results show that TPL promotes the expression of BACH1 protein in HKC cells,while inhibiting the expression of antioxidant target genes HMOX1 which located downstream of NRF2;qRT-PCR found that TPL promotes abnormal expression of mitochondrial-related genes.Conclusion:1.Triptolide induces kidney injury in mice and activates the cGAS-STING signaling pathway.2.Triptolide promotes the activation of cGAS-STING signaling pathway in human renal tubular epithelial cells.3.Triptolide may activate cGAS-STING signal pathway by damaging mitochondrial DNA. |
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