Study On The Pharmacodynamic Material Basis Of Saposhnikovia Divaricata(Turcz.) Schischk On Rheumatoid Arthritis And Its Accompanying Depression

Sposhnikovia divaricata（Turcz.）Schischk（S.divaricata）is the dried root of the unexhausted stem plant of the Umbelliferae.As China’s traditional Chinese medicine,S.divaricata is widely distributed in Northeast China,Inner Mongolia and other places.In the theory of traditional Chinese medicine,it belongs to surface relieving drugs.It is mainly used for headache and cold,rheumatism and arthralgia,rubella and itching,tetanus and other diseases.S.divaricata is widely used in the treatment of“Bi”syndrome,especially rheumatoid arthritis（RA）under the category of“Bi”syndrome,but its specific pharmacodynamic material basis and mechanism of action are still unclear.Modern chemical and pharmacological studies of traditional Chinese medicine have shown that S.divaricata mainly contains active ingredients such as chromone,coumarin,volatile oil,polysaccharide,etc.,and has pharmacological activities such as antipyretic,analgesic,anti-inflammatory,anti-tumor,immune regulation and neuroprotection.The previous research results of the research group showed that chemical components such as panaxynol and 4’-O-β-glucopyranosyl-5-O-methylvisamminol in S.divaricata showed anti-liver injury and neuroprotective effects through anti-inflammatory.On the basis of previous research,this paper prepared and analyzed the effective substances in S.divaricata,combined with pharmacological research,to further clarify the pharmacodynamic material basis and mechanism of S.divaricata’s prevention and treatment of RA and its accompanying depression.The specific experimental design and results are as follows:1.Preparation and structural characterization of S.divaricata active ingredientsFirstly,using silica gel column chromatography and preparative liquid phase technology,the ethanolic extract of S.divaricata was separated to obtain 7 kinds of small molecular compounds.After identification by high performance liquid chromatography,structural analysis by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry,it was confirmed that the above compounds belong to the chromones:cimifugin（Compound A,purity98.70%）,sec-O-glucosylhamaudol（Compound B,98.22%pure）,4’-O-β-glucopyranosyl-5-O-gethylvisamminol（compound C,99.75%pure）,prim-O-glucosylcimifugin（compound D,98.04%pure）.Coumarins:imperatorin（compound E,99.93%）,xanthotoxin（compound F,98.81%）.Volatile oil:panaxynol（compound G,purity 98.31%）.The crude polysaccharide of S.divaricata was obtained by hot soaking method and graded alcohol precipitation method with alcohol concentration ranging from 30%to 85%.After impurity removal,DEAE-52 cellulose and Sephadex G-100 column chromatography were separated and purified to obtain purified polysaccharides of each fraction.The total sugar content,protein,uronic acid and molecular weight of each fraction of polysaccharides were detected.At the same time,the biological activity of the purified polysaccharides of each fraction was detected,and it was found that the polysaccharide SD-60 was significantly stronger than other polysaccharide components in inhibiting the viability of fibroblast synovial cells（RA-FLS）.Therefore,the structure analysis of polysaccharide SD-60 was focused on.The monosaccharide composition analysis results showed that SD-60 was mainly composed of mannose（Man）,glucuronic acid（Glu A）,galacturonic acid（Gal A）,glucose（Glu）,galactose（Gal）and arabinose（Ara）.,and its molar ratio（%）is Man:Glu A:Gal A:Glu:Gal:Ara=4.16:1.97:37.46:2.12:16.42:37.87.The structural characteristics of SD-60 were further analyzed by comprehensive analysis methods such as infrared spectroscopy,NMR spectroscopy,methylation combined with GC-MS,scanning electron microscopy and Congo red experiment.The results show that SD-60is an acidic polysaccharide with triple helix structure,and its main chain is→{[4)-α-D-Gal Ap-（1→5）-α-L-Araf-（1→4）-α-D-Gal Ap-（1→5）-α-L-Araf-（1→6）-β-D-Galp-（1]₈→3,6）-α-D-Manp-（1→3）-β-D-Manp-（1→4,6）-β-D-Glcp-(1}₂₃→,at monosaccharide residues→3,6)-α-D-Manp-（1→and→4,6）-β-D-Glcp-（1→are connected at C6 and C4 respectively→5）-α-L-Araf-(1→branched chain;terminal chain T-β-D-Galp-(1→and T-β-D-Glc Ap-(1→linked at C5 of the branch.2.Research on the pharmacodynamic material basis of S.divaricata on collagen-induced rheumatoid arthritis（CIA）in rats.The CIA rat model was established,the fibroblasts（RA-FLS）of the CIA rats were isolated and cultured,the pharmacodynamic substances with significant effects on RA-FLS were screened,and the mechanism of action was elucidated.Then study the efficacy and mechanism of effective substances on CIA rats.（1）The effect and mechanism of S.divaricata components on the activity of RA-FLS.Panaxynol（PAL）and polysaccharide SD-60,which have a significant effect on the viability of RA-FLS cells,were screened by CCK-8 method from 7 small molecule compounds and 6purified polysaccharides in S.divaricata.Flow cytometry and Ed U proliferation experiments showed that PAL（40,50,60μM）and SD-60（30,40,50μg/m L）significantly induced apoptosis and inhibited proliferation of RA-FLS.Western blot results showed that PAL and SD-60significantly promoted Bax and Cleaved-caspase 3 and inhibited the protein expression of Bcl-2.Cell scratch and Transwell experiments showed that PAL（1,2,5μM）and SD-60（1,2.5,5μg/m L）significantly inhibited the migration and invasion of RA-FLS,and significantly inhibited MMP-2/9,Promotes protein expression of TIMP-1/2.The results of ELISA and Western blot experiments showed that PAL（1,2,5μM）and SD-60（1,2.5,5μg/m L）could inhibit the secretion of inflammatory factors and inhibit the signaling of TLR4/TRAF6,NF-κB/IκB-αactivation of the pathway.（2）Pharmacological activity and mechanism of PAL and SD-60 on CIA ratsAfter the successful establishment of the CIA rat model,PAL（40,80 mg/kg）and SD-60（40,80 mg/kg）were gavaged for 21 days,respectively.The experimental results show that both PAL and SD-60 can significantly reduce the arthritis index and immune organ index in CIA rats,and also have a significant inhibitory effect on inflammatory factors.H&E and Safranin O-fast green staining showed that PAL and SD-60 significantly relieved the inflammatory infiltration of rat knee and ankle joints,and also had a protective effect on cartilage.Western blot results showed that PAL and SD-60 significantly inhibited the activation of TLR4/TRAF6 and NF-κB/IκB-αsignaling pathways in the synovium of CIA rats.In conclusion,it is concluded that PAL and SD-60 are the main pharmacodynamic material basis of RA treatment.PAL and SD-60 have significant curative effect on RA in CIA rats,mainly by inhibiting Bcl-2,MMP-2/9,and promoting Bax,Cleaved-caspase 3,TIMP-1/2 protein expression.To achieve the purpose of inducing RA-FLS apoptosis and inhibiting its proliferation,migration and invasion.At the same time,by inhibiting the activation of TLR4/TRAF6 and NF-κB/IκB-αsignaling pathways,it can effectively alleviate the inflammatory infiltration of knee and ankle joints and the occurrence of synovial inflammation in CIA rats.3.Pharmacological effects and mechanism of S.divaricata on depressionAs a complication of RA,depression is very closely related to it.Therefore,further research was conducted on the effects of various components of the S.divaricata on depression.Depression induced by both inflammatory bursts and hyperactivation of the hypothalamic-pituitary-adrenal axis（HPA axis）is mainly explored.The antidepressant pharmacological activity and mechanism of each component of S.divaricata were studied by establishing LPS and CUMS-induced depression models.（1）Effects on LPS-induced mouse model of depressionFirst,the antidepressant effects of seven small molecules and six purified polysaccharides of S.divaricata in a mouse model of behavioral despair were investigated by tail suspension test（TST）and forced swim test（FST）.The experimental results show that PAL（0.1,1 mg/kg,intraperitoneal injection）can significantly reduce the immobility time of mice after 7 days of administration.Then,mainly investigated the antidepressant effect and mechanism of PAL.In the behavioral test results of LPS-induced mouse depression model,PAL significantly reduced the immobility time of TST and FST,and increased the movement time and exploration of mice in the open field test（OFT）and elevated plus maze test（EPM）.It also significantly improved the depression-like behavior of mice,and inhibited the secretion of inflammatory factors and oxidative stress;inhibited the expression of NF-κB/IκB-αand promoted the protein expression of BDNF/Trk B in the hippocampus of mice.（2）Effects on LPS-induced BV-2 microglial inflammation modelBV-2 microglia are mainly found in the hippocampus,and their hyperactivation is a key factor in inflammation-induced depression.The experimental results showed that PAL（1,5,10μM）could inhibit the viability of BV-2 microglial cells,inhibit the secretion of inflammatory factors and the occurrence of oxidative stress in cells,inhibit NF-κB/IκB-αin cells,and promote BDNF/Trk B protein expression.（3）Effects on chronic unpredictable mild stress（CUMS）induced mouse model of depressionHyperactivation of the HPA axis is a major factor in CUMS induced depression in mice.The results of behavioral experiments showed that PAL（0.1,1 mg/kg,intraperitoneal injection）could effectively improve the depressive state of mice after 21 days of treatment.Inhibit the secretion of corticotropin-releasing hormone（CRH）,adrenocorticotropic hormone（ACTH）and corticosterone（CORT）in the HPA axis;promote the expression of 5-HT/5-HIAA and DA/HVA in the hippocampus,enhance the Metabolism of neurotrophic factor（BDNF）,proteins that promote glutamate receptor 1（Glu R1）,postsynaptic dense protein 95（PSD95）,Synapsin I,and hypothalamic glutathione reductase（GR）expression to improve synaptic function.In conclusion,It is concluded that PAL is the main pharmacodynamic material basis for the treatment of depression by S.divaricata.Compared with other components of S.divaricata,it was found that the main pharmacodynamic material basis of S.divaricata in the treatment of RA was PAL and SD-60,and the main pharmacodynamic material basis for the treatment of RA complicated with depression was PAL.In addition,PAL is a volatile oil,which is in line with the pungent temperature of S.divaricata and the nature and taste of episodic drugs.In addition,there are many water decoctions for traditional S.divaricata medicine,while polysaccharide SD-60 is very soluble in water,which is also similar to the medicine concept of windbreak.The above research provides new ideas for the quality control,processing and development and utilization of traditional Chinese medicine S.divaricata,and also lays a foundation for the development of therapeutic drugs for RA and its accompanying depression.4.The effect and mechanism of PAL on depression in CIA ratsFirst,the CIA rat model was established,and then the rats with obvious depressive symptoms were screened by TST as the RA-complicated depression model animals.PAL（0.07,0.7 mg/kg,intraperitoneal injection）was administered for 7 days and related indicators were measured.The results of TST test showed that PAL significantly reduced the immobility time of rats.Inflammatory factors in the rat serum and oxidative stress in the hippocampus were significantly inhibited,the hyperactivation of the HPA axis was inhibited,and the metabolic capacity of neurotrophic factors was enhanced.The results of Western blot experiments showed that PAL inhibited the activation of the NF-κB/IκB-αsignaling pathway,enhanced the protein expressions of BDNF/Trk B,Glu R1,PSD95 and Synapsin I,and effectively improved the synaptic transmission function in the rat hippocampus.All the above experimental results show that the main pharmacodynamic material basis of S.divaricata in the treatment of RA is PAL and SD-60,and the main pharmacodynamic material basis of S.divaricata in the treatment of RA-complicated depression is PAL.PAL belongs to volatile oil,which is in line with the drug theory of S.divaricata as a traditional Chinese medicine with“Xin Wen”and the exterior syndromes drugs.The traditional medicine of S.divaricata is mostly decoction,and the polysaccharide SD-60 is easily soluble in water,which is also consistent with the traditional medicine concept of S.divaricata.Through the above research,we hope to provide new ideas for the quality control,processing,development and utilization of traditional Chinese medicine S.divaricata,and lay a foundation for the research and development of therapeutic drugs for RA and associated depression.