To Explore The Analgesic Mechanism Of Danggui Sini Decoction Cold Medicine And Its Components Based On TRP Signal Pathway Regulated By Artemin/GFR?3

Purpose:Danggui Sini Decoction is a prescription for treating cold deficiency in the past.It is now widely used to treat various neuropathic pain caused by cold.Previous studies have confirmed that Danggui Sini Decoction can comprehensively inhibit temperature-sensitive TRP channels and treat sciatic nerve injury caused by CCI.It can not only inhibit cold pain,but also thermal pain and mechanical pain.This paper will further explore the analgesic effect and mechanism of the Tetrapanax papyriferus and Paeonia lactiflora,in order to reveal the scientific and rationality of these drugs for analgesia.Methods:1.Sixty-four SD rats were randomly divided into eight groups according to their weight,sham operation group,model group,pregabalin group,mecobalamin group,Tetrapanax papyriferus group,Hederagenine group,Paeonia lactiflora group and Paeoniflorin group.Except for the sciatic nerve exposed in the sham group,all other rats received CCI ligation.Rats in the sham operation group were intragastrically administered with normal saline every day,and the pregabalin group was intragastrically administered with 10mg/kg·d·bid pregabalin,and the other rats were intragastrically administered with 15mg/kg·d·bid mecobalamin,0.6356g/kg·d·bid Tetrapanax,10mg/kg·d·bid Hederagenine,0.9535g/kg·d·bid paeony,and 10mg/kg·d·bid paeoniflorin respectively.Rats were subjected to acetone test,hot plate test and Von Frey test in1 day before surgery,1,4,7,14,21 day after surgery.On the 21st day after operation,after the test of behavior,blood was collected from the abdominal aorta after anesthesia,and the sciatic nerve and L4-6 DRG neurons were removed from the ligation site.Elisa was used to detect the levels of IL6,IL1?,and TNF-?in serum.Western-blot was used to detect the expression of Artemin and TRP protein in DRG neurons.The expressions of Artemin,GFR?3,TRPV2 and TRPV3 and Immunofluorescence of TRPA1,TRPV1,GFR?3 in DRG neurons were carried.2.CCK8 was used to detect the effect of paeoniflorin and H₂O₂ on the activity of Schwann cells.Based on screening appropriate paeoniflorin concentration and H₂O₂ injury concentration,the experiment was divided into normal group?control?,model group?model?,p38 inhibitor group?SB203580?,paeoniflorin 50?M group?PF 50?and paeoniflorin 100?M group?PF 100?.SB203580 and paeoniflorin were pretreated 24 h before H₂O₂ treatment.All groups except the normal group were treated with 200?M H₂O₂ serum-free medium for 4 h.The ROS activity of Schwann cells in each group was detected by ROS reactive oxygen species,the apoptosis of Schwann cells was observed by Hoest33258,the apoptosis of Schwann cells was detected by flow cytometry,Bcl-2 and Bcl-xl m RNA levels were detected by PCR,and apoptotic proteins and TRP proteins were checked by Western-blot.Result:1.Compared with the sham operation group,CCI rats showed obvious thermal hyperalgesia,mechanical hyperalgesia and cold hyperalgesia from 4 days after surgery?4,7,14,21d,P<0.05?.Compared with the model group,all drugs including pregabalin and mecobalamin can alleviate thermal,mechanical and cold hyperalgesia in CCI rats starting on the 7th day?7,14,21d,P<0.05?,Pregabalin,mecobalamin,and Tetrapanax alleviated the cold pain sensitivity of CCI rats on the 4th day after surgery?P<0.05?.Compared with the sham operation group,except that the rats in the pregabalin group could return to normal levels 21 days after the operation,the rest of the rats could not be sensitive to cold pain at 4,7,14,and 21 days after the operation.Thermal hyperalgesia and mechanical hyperalgesia returned to normal levels?4,7,14,21 days,P<0.05?.2.Compared with serum inflammatory factor levels in sham-operated rats,serum levels of IL6,IL1?,and TNF-?in CCI rats increased significantly?P<0.05?.3.Compared with the inflammation level of CCI rats in the model group,pregabalin,mecobalamin,Tetrapanax and Paeonia lactiflora can reduce the levels of IL6 and IL1??P<0.05?,and all drugs can reduce the TNF-??P<0.05?.4.21 days after CCI,the expression levels of Artemin,TRPV1,TRPV4,TRPA1,TRPM8,and p-p38MAPK increased in the model group,and the m RNA expression levels of Artemin,GFR?3,TRPV2,and TRPV3 increased,and the fluorescence intensity of TRPA1,TRPV1,and GFR?3increased.?P<0.05?.5.Compared with the model group,all drugs are capable of reducing the raised Artemin,TRPV1,TRPV4,TRPA1,TRPM8,and p-p38MAPK?P<0.05?;reducing the expressions of m RNA of Artemin,GFR?3,TRPV2,TRPV3,and reducing TRPA1,TRPV1,GFR?3 fluorescence intensity.6.Paeoniflorin can protect Schwann cells from H₂O₂-induced increase in ROS and nuclear apoptosis.7.Pretreatment of paeoniflorin and SB203580 can protect Schwann cells from H₂O₂-induced apoptosis.8.In the model group,the expression levels of anti-apoptotic proteins Bcl-2,Bcl-xl and their m RNAs decreased,the expressions of pro-apoptotic proteins caspase3,c-caspase3,c-caspase7increased,and the expression of p-p38MAPK increased.9.SB203580 and paeoniflorin can increase the expression levels of Bcl-2,Bcl-xl and their m RNA,reduce the levels of caspase3,c-caspase3,c-caspase7,and decrease the expression of p-p38MAPK,TRPA1,TRPV1.In conclusion:1.Artemin,GFR?3,TRPA1,TRPV1-4,TRPM8,and p-p38MAPK are closely related to neuropathic pain.2.Paeonia tactilora,Tetrapanax,Paeoniflorin and Hederagenine can alleviate thermal hyperalgesia,mechanical hyperalgesia and cold hyperalgesia in CCI rats and can promote the repair of injured sciatic nerve.3.Paeonia tactilora,Tetrapanax,Paeoniflorin and Hederagenine can inhibit phosphorylation of p38MAPK,and thus inhibit downstream TRP protein;4.Paeonia tactilora,Tetrapanax,Paeoniflorin and Hederagenine can inhibit the expressions of inflammatory factors,Artemin,and GFR?3,and then inhibit p38MAPK phosphorylation;5.Paeoniflorin can regulate downstream apoptotic proteins and anti-apoptotic proteins by inhibiting p38MAPK phosphorylation,thereby protecting Schwann cells from H₂O₂-induced damage and apoptosis.6.Paeonia tactilora,Tetrapanax,Paeoniflorin and Hederagenine regulate TRPs by inhibiting the expressions of Artemin/GFR?3 signaling pathway and inflammatory factors.