| Objective:The neurovascular unit and the Notch signaling pathway were used as the starting point for research,this study explored therapeutic efficacy and regulation targets of the classic hour unit combination treatment plan in Tibetan Medicine(Seventy Flavor Pearl Pills,Ruyi Zhenbao Pills and Ershiwei Chenxiang Pills were taken in the early morning,brunch and late evening)on rats with brain cerebral ischemia-reperfusion.The purpose of research was to provide experimental data on modern biology for characteristics therapy which was applied for the treatment of Longzhibu disease based on traditional Tibetan medical theory and clinical experience.Methods:By methods of literature collection,this study explored the theoretical basis on the pathogeny,diagnosis and treatment of Longzhibu disease in order to make investigation on theoretical foundation and guiding principles to select reasonable time for taking drugs in Tibetan medicine.Based on the previous data mining research,this experimental research on therapeutic efficacy and regulation targets of the classic hour unit combination treatment plan(Seventy Flavor Pearl Pills,Ruyi Zhenbao Pills and Ershiwei Chenxiang Pills were taken in the early morning,brunch and late evening)was conducted.The models of focal cerebral ischemia and other rats were randomly and equally divided into six groups:sham operation group(S group),model group(M group),positive control group(P group),Seventy Flavor Pearl Pills group(SF group),two medicine combination group(TM group),and three medicine combination group(TC group).Each group was separated into four subgroups according to ischemia reperfusion time including 1 days,3 days,7 days and 15 days.With the neurovascular unit and Notch signal pathway serving as the direction of research,a variety of modern experimental methods such as neuroethology testing,histopathological staining testing,peripheral blood status testing and Western blot testing were applied to investigate moderating role of the classic hour unit combination treatment plan on the restoration of nerve and limb function,hemorheology changes,and the protein expression of Ang-1,Notch1,Jagged 1 in the hippocampus of the brain,which interpreting the therapeutic efficacy and regulatory mechanism of the treatment for Longzhibu disease.Result:(1)The Longzhibu disease was caused by the increase,reduction or disorders of the rlung,mkhris-pa and bad-ka under the influence of external environment including the changes of weather,seasons,day-night cycle,regions and foods as well as internal environment including strength changes and emotional distractions,finally the fundamental changes of the rlung,mkhris-pa and bad-ka broke the balance of body.Due to the instability and imbalance changes on quantity and position of the three major factors,the related characteristics and functions became abnormal with causing pathological damage in many aspects,and finally brought about outstanding correlation symptoms with rlung,blood and pulse.The Longzhibu disease had a certain circadian rhythm,that was more likely to develop and change in the period of dusk until dawn.According to the specific content of Tibetan calendar system and concept of wholism,the combination of polypharmacy and chrono-medication plan(Seventy Flavor Pearl Pills,Ruyi Zhenbao Pills and Ershiwei Chenxiang Pills were taken in the early morning,brunch and late evening)was applied in the overall medical treatment of the Longzhibu disease in accordance with the time rhythm,drug properties and disease conditions,especially combined with the particular symptoms of the Longzhibu disease.(2)?The behavioral results of animal experiments shower that S group had notable differences in behavioral scores compared with other model groups(P<0.05).The result of beam-walking test and screen test at 1d and 3d besides limb placement test at 1d showed that there were no obvious differences between the various model groups(P>0.05).The result of beam-walking test and limb placement test at 7d and 15d showed that SF group,TM group,and TC group had significant differences compared with M group at the same time(P<0.05).In particular,TC group had a statistical difference compared with P group(P<0.05).The limb placement test of TC group at 7d was better than that of SF group at the same time(P<0.05).The result of screen test at 7d and 15d showed that TC group had notable differences with M group(P<0.05).From the comparison results of the same group at different times,the research showed that TC group at 7d and SF group,TM group,and TC group at 15d each had a remarkable difference in the contrast of the two time points of 1d and 3d in terms of beam-walking test and limb placement test(P<0.05).?The results of HE staining showed that clear ischemic region and penumbra appeared in M group with obvious nuclear contraction and softening lesion.At 7d and 15d,arrangement of brain tissue in M group manifested a slight improvement,TM group and TC group obtained significant improvements in nuclear morphology and softening focus compared with M group at the same time.From the comparison results of the same group at different times,morphological features of neuron and neuron edema were significantly relieved at 7d and 15d in SF group,TM group and TC group.?The results of blood rheology detection showed that the hemorheological indexes of each model group had risen and manifested significant differences compared with S group(P<0.05).There were no obvious differences between the various model groups at 1d(P>0.05).Hematocrit,red blood volume width coefficient variation,and mean platelet volume in TC group were significantly lower than those of M group at 3d(P<0.05).SF group,TM group,and TC group all had notable reductions in hematocrit,red blood volume width variation coefficient variation,and platelet distribution width compared with M group at 7d and 15d(P<0.05).The hematocrit,red blood volume width coefficient variation,and platelet distribution width of TC group were remarkable lower than those of P group(P<0.05).The hematocrit of TC group was lower than that of P group(P<0.05).The platelet distribution width of SF group,TM group,and TC group all got significantly decrease compared with M group at 15d(P<0.05).From the comparison results of the same group at different times,the hemorheology index of each M group appeared a trend of declining after increasing,and reached the highest value at 3d(P<0.05).SF group,TM group,and TC group all manifested remarkable differences in hematocrit and mean platelet volume at 7d and 15d compared with those of 3d(P<0.05).There was a remarkable difference in the red blood cell width coefficient variation of SF group,TM group,TC group at 7d compared with those of 3d,TC group at 15d showed the same distinction as well(P<0.05).Notable differences were also occurred in platelet distribution width between model groups at 15d compared with those of 3d(P<0.05).?The results of western blot showed that the protein expressions of Ang-1,Notchl and Jaggedl increased,but there was no significant difference between the various model groups at Id(P>0.05).The expressions of Ang-1,Notch1,and Jaggedl proteins in TC group were enhanced only compared with P group,M group and S group separately at 3d(P<0.05).Ang-1,Notchl,and Jaggedl proteins expression of TC group were higher than those of S group and M group with statistica differences at 7d and 15d(P<0.05).The protein expressions of Ang-1,Notchl and Jaggedl of TC group were obviously higher than that of SF group at 15(P<0.05).Conclusion:Seventy Flavor Pearl Pills and Ruyi Zhenbao Pills were selected to regulate rlung,blood and veins flow in the early morning and brunch,while Seventy Flavor Pearl Pills and Ershiwei Chenxiang Pills were taken in the early morning and the late evening to regulate rlung in the classic hour unit combination treatment plan of Tibetan Medicine.That plan not only fitted the variation tendency of the three major factors and the time pattern of the disease transition cycle,but also followed the trend of stomach fire to promote the absorption and metabolism of drugs,and achieved the purpose of effectively correcting the imbalance and disorder of the three major factors.Further experimental results indicated that the treatment plan reduced the HCT,RD W,MPV and PD W of peripheral blood and raised protein expressions of Ang-1,Notch1,and Jagged1 in hippocampus to improved nerves activity and the function of limbs.It was confirmed that the hour unit combination treatment plan of Tibetan medicine had a positive therapeutic effect on multiple targets of rats with brain cerebral ischemia-reperfusion.However,the effects of activating Notch signaling pathway and protecting neurovascular units was worthy of further in-depth study. |
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