Pharmacodynamic Studies Of Hippophae Rhamnoides L. On Nervous System Related Diseases

As people's lifestyle and dietary patterns change,the pace of life accelerates,as well as the living environment deteriorates,the incidence of neurological-related diseases such as fatigue,stroke,depression and hypoxia is gradually increasing.These disease seriously affecting people's health,quality of life and longevity.Therefore,there is an urgent need to develop a medicinal food or adjuvant having an effective low toxicity against diseases of the nervous system.Hippophae rhamnoides L.is a traditional medicinal food with Chinese characteristics and has a variety of pharmacodynamic activities.The Tibetan Pharmacopoeia "Crystal Herbs" has a related role in regulating the nervous system such as refreshing and anti-hypoxia.Polysaccharide is the most abundant component in H.rhamnoides.At present,there is no systematic research report on the role of H.rhamnoides.polysaccharide in regulating nervous system.In this study,H.rhamnoides.polysaccharides were isolated and the neuroprotective effects,as well as mechanisms of its fractions were discussed.The results may promote their in-depth development and application in related clinical medicine.Firstly,the water-soluble polysaccharides from H.rhamnoides,were isolated,purified and performed to do the composition analysis according to the method established by the laboratory of Key Laboratory of Tibetan Medicine Research,Chinese Academy of Sciences.The water extract,alcohol precipitation and DEAE-cellulose ion exchange chromatography were used to obtain total HRWP.The HRWP was further fractionated by DEAE-cellulose ion exchange column to obtain HRWP-N and HRWP-A.HRWP-N is a neutral HG-type pectin,while HRWP-A is a highly methylated acidic HG-type pectin fraction.HRWP,HRWP-N and HRWP-A were administered to mice to investigate the neurological diseases related to depression,anti-brain tumor and anti-stroke,and the active units with better activity were compared.In this study,effect of H.rhamnoides polysaccharide on antidepressant-like diesease through OFT,FST and CUMS model.Firstly,the FST model was used to determine whether HRWP had antidepressant effect,the water-soluble polysaccharides isolated from H.rhamnoides,L.barbarum,L.ruthenicum and N.tangutorum were also evaluated at the same time.Then FST was used to further analyze the active fraction of HRWP with better antidepressant-like effect.The results showed that the HRWP could reduce the immobile time of mice,and the effect was not related to nerve excitation.At the same time,the polysaccharide had no significant effect on the weight of FST mice,but it had a recovery effect on the liver weight reduction caused by depression-like.Comparing to the HRWP-N,HRWP-A significantly increased the immobilization time of FST mice.Further,the effects of HRWP-A on the recovery of depression model mice were discussed through the OFT and CUMS,and the effects of HRWP-A on neurotransmitters and oxidative stress molecules in CUMS mice were analyzed.The results shown that HRWP-A can inhibit the depression-like in CUMS model.At the same time,HRWP inhibited the reduction of FST-induced glucose(GLU),superoxide dismutase(SOD)and glutathione peroxidase(GPx),creatine phosphate kinase(CK),lactate dehydrogenase(LDH),while increased triglycerides(TG)and malonaldehyde(MDA).ELISA results showed that HRWP-A increased the levels of CUMS mouse neurotransmitter serotonin(5-HT),dopamine(DA).This process was accompanied by increasing level of BDNF and pCREB/CREB in hippocampus.These results suggested that the mechanism of HRWP-A antidepressant effect may involve the anti-oxidative effect and maintenance of neurotransmitter homeostasis in the brain and the upregulation of BDNF-CREB pathway.The current study was designed to explore the protective effect of HRWP-A on middle cerebral artery occlusion(MCAO)-induced focal cerebral ischemia in mice and the underlying mechanisms.C57BL/6 mice received HRWP-A(50,100 and 200 mg/kg)i.g.for 14 days and then were subjected to occlusion of the right middle cerebral artery followed by 2 additional days of HRWP-A administration.Brain infarct size,behaviour test including the neurological severity score(mNSS),the corner test and the fore limb placement test,brain tisssue analyses and ELISA were performed to assess neuroprotetive effect of HRWP-A in these mice.The results showed that HRWP-A significantly reduced the infarct size and improved the neurological dysfunction in MCAO mice.HRWP-A improved the neuroprotective status,as it blocked the increase in malondialdehyde(MDA)and NO(nitric oxide)while attenuating the decrease in dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)levels in the brain,as well as attenuating the pro-inflammatory indictors of TNF-?,IL-1? and IL-6 levels in serum.This process accompanied with the downregulation of NLRP3 and MMP9 expression while upregulation of claudin-5 expression as well.Altogether,HRWP-A induced an improvement in brain impairment.The neuroprotective nature of HRWP-A provides evidence for its proposed application in functional foods for potential neuroprotection.Direct cytotoxic effect of HRWP-A on Lewis(LLC1),human giloma U87,mouse giloma GL261 and human breast tumor(231)cells was observed.Results shown that at all dosages,HRWP-A has no direct cytotoxicity against these cells.An antitumor activity assay showed that HRWP-A could significantly inhibit the Lewis lung carcinoma(LLC)growth in tumor-bearing mice.Then,the GL-261-tumor bearing model was used to analysis the antitumor effect of HRWP-A.The results show that HRWP-A can prolong the survival of tumor-bearing mice.The number of immune cells in the tumor microenvironment in situ was analyzed by mass cytometry.The results showed that the number of monocytes in the tumor microenvironment in HRWP-A group significantly increased compared with the model group.The number of Treg,CD8 + T,CD4 + T,and cDC was no significant changing between HRWP-A group and model group in the tumor situ.When mononuclear cells arrived in the tissue,they would differentiated into macrophages.So,we further discussed whether HRWP-A had a direct effect on mouse peritoneal macrophages.In addition,we investigated the possible mechanism underlying the effects of HRWP-A on mouse peritoneal macrophages.qPCR and western blot revealed that HRWP-A upregulated the expression of TLR4 mRNA in vitro.This process was accompanied by a clear increase in MyD88 expression and p-I?B-?,but these effects were largely abrogated by pretreatment with anti-TLR4 antibodies.The effects of HRWP-A on macrophage NO,IL-1? and IL-6 production were also inhibited by anti-TLR4 antibodies and were greatly influenced by the NF-?B inhibitor PDTC.Moreover,HRWP-A failed to induce the production of NO,IL-1? and IL-6 in peritoneal macrophages prepared from C3H/HeJ mice,which have a point mutation in the Tlr4 gene,suggesting the involvement of the TLR4 molecule in HRWP-A-mediated macrophage activation.These results may have important implications for our understanding of the structure-activity relationship of immunopotentiating polysaccharides from medicinal herbs.In this study,we screened the active compounds of H.rhamnoides to explore its role in neuromodulation on nervous system diseases.The study may lay the foundation for the development of new drugs or auxiliary drugs for neurological diseases.At the same time,by studying the possible mechanism of polysaccharide on the protection mechanism of the nervous system is helpful to understand the pathogenesis and influencing factors of nervous system diseases.