| Objective:1.It is clear that the antidepressant effect of Baihe Dihuang decoction(Zhongjing original prescription)is related to improving the dysfunction of GABAergic intermediate neurons in prefrontal cortex2.To clarify the pharmacological mechanism of Baihe Dihuang Decoction(Zhongjing original prescription)by reducing the expression of mi RNA-144-3p in prefrontal cortex,increasing the expression of GAD 67 and VGAT,affecting the synthesis andtransport of GABA before synapse,increasing the release of GABA,correcting the excitation/inhibition imbalance of central neurons,and then alleviating the depressivesymptoms.Methods:Part I: Study on the fingerprint of Lily Dihuang Decoction(Zhongjing original prescription)freeze-dried powder.Take 200 ul of Lily Dihuang Decoction(Zhongjing original prescription)freeze-dried powder solution,add 1 ml of 80% methanol solution,and vortex the sample for 10 min.Centrifuge at 4℃ for 10 min with a centrifugal force of20000 xg,and then take the supernatant,filter it and analyze it on the computer.Part Ⅱ:Study on the antidepressant mechanism of Baihe Dihuang decoction(Zhongjing original prescription)regulating the activity of GABAergic neurons in prefrontal cortex.Sixty SPF C57BL/6 male mice were randomly divided into blank group,model group,positive drug group and high,middle and low dose groups of Lily Dihuang decoction,with 10 mice in each group.Chronic unpredictable mild stress(CUMS)was used to establish a depression model,which lasted for four weeks.After one week of modeling,the corresponding drugs were given to the stomach for three weeks respectively.After the end of modeling,the construction of behavioral test model and the improvement of symptoms after the administration of Baihe Dihuang Decoction.The expression of neurotransmitters and inflammatory factors in serum of mice was detected by ELISA.QRT-PCR was used to detect the m RNA expressions of Gad67,VGAT and GAT-3 in the prefrontal cortex of mice and the expression of mi RNA-144-3p.Western Blot was used to detect the protein expressions of Gad67,VGAT and GAT-3 in the prefrontal cortex of mice.Part Ⅲ:Antagonizing the activity of GABAergic neurons in prefrontal cortex GAB A slowing down the effect of Lily Dihuang Decoction(Zhongjing original prescripti on)on improving depression-like behavior in mice.Fifty SPF C57BL/6 male mice were divided into Control+p AV-CMV-GFP-mi RNA-NC group,CUMS+p AV-CMV-GF P-mi RNA-NC group,CUMS+p AV-CMV-GFP-mi RNA-NC+LBRD group and CUMS+p AV-GAD67-GFP-mi RNA-144-3p group,Control+p AV-Gad67-GFP-mi RNA-144-3p gro up,10 mouses in each group,were injected with adeno-associated virus and blank virus expressing mi RNA-144-3p in the midbrain of the prefrontal cortex of mice,re spectively.After the model was successfully constructed,they were given correspon ding drug treatment.The construction of behavioral test model and the improvementof symptoms after the administration of Baihe Dihuang Decoction.The expression of neurotransmitters and inflammatory factors in serum of mice was detected by EL ISA.QRT-PCR was used to detect the m RNA expression of Gad67 and VGAT in t he prefrontal cortex of mice.Result:Part I: Study on fingerprint of Lily Dihuang Decoction freeze-dried powder.The results of ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry showed that 489 compounds in Lily Dihuang decoction freeze-dried powder scored more than 60 points in the best matching of mz Cloud database.A total of 31 compounds were identified,including adenosine,aloperine,pyridoxal,berberine,choloside-3-acrylic acid,catalpol,ferulic acid,phenolic glycoside A,guanine and so on.PartⅡ: Study on the antidepressant mechanism of Baihe Dihuang decoction(Zhongjing original prescription)regulating the activity of GABAergic neurons in prefrontal cortex.Compared with the blank group,the intake of sugar water in the model group decreased significantly(P<0.01).After drug intervention,compared with the model group,the sugar water intake of mice in the high and middle dose groups of Baihe Dihuang Decoction recovered(P<0.05,P<0.01),and the sugar water intake in the positive drug group also increased significantly(P<0.01).The results of open field experiment showed that compared with the blank group,the exploration time in the central area of the model group was significantly reduced(P<0.01).After drug intervention,compared with the model group,the mice in the high,middle and low dose groups of Baihe Dihuang Decoction explored the central area significantly(all P<0.01),and the mice in the positive drug group explored the central area significantly(P<0.01).The results of forced swimming experiment showed that compared with the blank group,the immobility time of mice in the model group increased significantly(P<0.01).After drug intervention,compared with the model group,the immobility time in the middle and low dose groups of Baihe Dihuang Decoction was significantly shortened(P<0.01,P<0.05),and the immobility time in the forced swimming experiment of mice in the positive drug group was significantly shortened(P<0.01).The experimental results of the cross-elevated maze showed that compared with the blank group,the exploration time of the model group mice in the open arm was significantly reduced(P<0.01).After drug intervention,compared with the model group,the exploration time of mice in the high,middle and low dose groups of Baihe Dihuang Decoction in the open arm was significantly improved(all P<0.01),and the exploration time of mice in the positive drug group was significantly improved(P<0.01).The results of water maze experiment showed that compared with the blank group,the mice in the model group exercised more aimlessly,and the times of crossing the platform were significantly reduced(P<0.01).After the intervention,compared with the model group,the times of crossing the platform in the high,middle and low dose groups of Baihe Dihuang Decoction increased significantly(P<0.01,P<0.01,P<0.05),while the times of crossing the platform in the positive drug group decreased significantly(P<0.01).The results of ELISA showed that compared with the blank group,the contents of 5-HT,GABA and DA in the serum of the model group were significantly decreased(P<0.01).The contents of IL-6 and TNF-α increased significantly(P<0.01).The content of IL-10 decreased significantly(P<0.01).Compared with the model group,the contents of 5-HT,GABA and DA in serum of mice in Baihe Dihuang Decoction(Zhongjing original prescription)group were significantly increased(P<0.01).The contents of IL-6 and TNF-αdecreased significantly(P<0.01).The content of IL-10 increased significantly(P<0.01).The results of q RT-PCR showed that compared with the blank group,the m RNA expressions of Gad67,VGAT and GAT-3 in the prefrontal lobe of the model group decreased significantly.Compared with the model group,the m RNA expressions of Gad67,VGAT and GAT-3 in the prefrontal tissue of mice in Baihe Dihuang Decoction(Zhongjing original prescription)group were significantly increased.Compared with the blank group,the expression of mi RNA-144-3p in the prefrontal tissue of the model group increased significantly.After the intervention of Baihe Dihuang Decoction,compared with the model group,the expression of mi RNA-144-3p in the prefrontal cortex of mice in Baihe Dihuang Decoction(Zhongjing original prescription)group obviously recovered.Western Blot results showed that compared with the blank group,the protein expressions of Gad67,VGAT and GAT-3 in the prefrontal lobe of the model group decreased significantly.Compared with the model group,the expression of Gad67,VGAT and GAT-3 protein in the prefrontal tissue of mice in Baihe Dihuang decoction(Zhongjing original prescription)group increased significantly.Part Ⅲ:Antagonizing the activity of GABAergic neurons in prefrontal cortex G ABA slowing down the effect of Lily Dihuang Decoction(Zhongjing original prescr iption)on improving depression-like behavior in mice.Compared with the Control+p AV-CMV-GFP-mi RNA-NC group,the sugar water intake of CUMS+p AV-CMV-GFP-mi RNA-NC group decreased significantly(P<0.01).Compared with CUMS+p AV-C MV-GFP-mi RNA-NC group,the sugar water intake of mice in CUMS+p AV-CMV-G FP-mi RNA-NC+LBRD group obviously recovered(P<0.01).Compared with the Con trol+p AV-CMV-GFP-mi RNA-NC group,the sugar water intake of the mice in the C ontrol+p AV-GAD67-GFP-mi RNA-144-3p group decreased significantly(P<0.01).Com pared with CUMS+p AV-CMV-GFP-mi RNA-NC+LBRD group,the sugar water intakeof mice in CUMS+p AV-GAD67-GFP-mi RNA-144-3p+LBRD group decreased signifi cantly(P<0.01).The results of open-field experiments showed that compared with t he Control+p AV-CMV-GFP-mi RNA-NC group,the mice in CUMS+p AV-CMV-GFPmi RNA-NC group had significantly shorter exploration time in the central area(P<0.01).Compared with CUMS+p AV-CMV-GFP-mi RNA-NC group,the mice in CUM S+p AV-CMV-GFP-mi RNA-NC+LBRD group had significantly longer exploration tim e in the central area(P<0.01).Compared with the Control+p AV-CMV-GFP-mi RNANC group,the mice in the control+p AV-GAD67-GFP-mi RNA-144-3p group spent si gnificantly less time exploring the central area(P<0.01).Compared with the CUMS+p AV-CMV-GFP-mi RNA+LBRD group,the mice in the CUMS+p AV-GAD67-GFP-m i RNA-144+LBRD-3p group spent significantly less time exploring the central area(P<0.01).The results of forced swimming experiment showed that the immobility ti me of mice in CUMS+p AV-CMV-GFP-mi RNA-NC group was significantly longer th an that in Control+p AV-CMV-GFP-mi RNA-NC group(P<0.01).Compared with CU MS+p AV-CMV-GFP-mi RNA-NC group,the immobility time of CUMS+p AV-CMV-G FP-mi RNA-NC+LBRD group was significantly shortened(P<0.01).Compared with Control+p AV-CMV-GFP-mi RNA-NCgroup,the immobility time of mice in Control+p AV-GAD67-GFP-mi RNA-144-3p group was significantly increased;Compared with CUMS+p AV-CMV-GFP-mi RNA-NC+LBRD group,the immobility time of mice in c ums+p AV-GAD67-GFP-mi RNA-144-3p+LBRD group was significantly shortened(p<0.01).The results of ELISA showed that the contents of serum GABA and 5-HT i n CUMS+p AV-CMV-GFP-mi RNA-NC group were significantly lower than those in Control+p AV-CMV-GFP-mi RNA-NC group(P<0.01).That is,compared with CUMS+p AV-CMV-GFP-mi RNA-NC group,the contents of serum GABA and 5-HT in CU MS+p AV-CMV-GFP-mi RNA+LBRD group were obviously recovered(P<0.01).Comp ared with the Control+p AV-CMV-GFP-mi RNA-NC group,the serum GABA and 5-H T in the Control+p AV-GAD67-GFP-mi RNA-144-3p group decreased significantly(P<0.01).Compared with CUMS+p AV-CMV-GFP-mi RNA+LBRD group,the contents ofserum GABA and 5-HT in cums+p AV-GAD67-GFP-mi RNA-144-3p+LBRD group d ecreased significantly(P<0.01).The results of q RT-PCR showed that compared withthe Control+p AV-CMV-GFP-mi RNA-NC group,the m RNA expression of Gad67 an d VGAT in the prefrontal cortex of mice in CUMS+p AV-CMV-GFP-mi RNA-NC gro up decreased significantly(P<0.01).Compared with the Control+p AV-CMV-GFP-mi R NA-NC group,the m RNA expression of Gad67 and VGAT in the prefrontal cortex of the Control+p AV-GAD67-GFP-mi RNA-144-3p group decreased significantly(P<0.01).Compared with CUMS+p AV-CMV-GFP-mi RNA-NC group,the m RNA expressio ns of Gad67 and VGAT in the prefrontal cortex of mice in CUMS+p AV-CMV-GFPmi RNA-NC+LBRD group recovered significantly(P<0.01).Compared with CUMS+p AV-CMV-GFP-mi RNA-NC+LBRD group,the m RNA expression of Gad67 and VGA T in the prefrontal cortex of mice in CUMS+p AV-GAD67-GFP-mi RNA-144-3p+LBR D group decreased significantly(p<0.01).Conclusion:1.Baihe Dihuang Decoction(Zhongjing original prescription)can reduce the level of peripheral and central inflammation,antagonize the excitement of HPA axis,correct the imbalance of excitatory/inhibitory neurotransmitters,reduce the damage of cortical neurons induced by stress,and improve depression-like behavior.Baihe Dihuang Decoction(Zhongjing original prescription)can reduce the expression of mi RNA-144-3p in prefrontal cortex,increase the expression of Gad67 and VGAT,affect the synthesis and transport of GABA before synapse,increase the release of GABA,enhance the correlation of synaptic transmission efficiency,restore the function of GABA neurons to receive coding and output information,and then alleviate the pharmacological mechanism of depression symptoms. |
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