The Study Of Yiqi Huoxue Recipe In The Treatment Of Cervical Spondylotic Myelopathy And Its Active Ingredients Nanoparticles For Repairing Spinal Cord Injury

Objective1.To evaluate the clinical efficacy of Yiqi Huoxue Recipe in the treatment of mild cervical spondylotic myelopathy(CSM)with Qi deficiency and blood stasis syndrome.2.To innovatively design and synthesize nanoparticles of the active ingredients from Chinese herbs of supplementing Qi and activating blood circulation under the guidance of compatibility theory of traditional Chinese medicine(TCM),and to study its effect and mechanism on the repair of spinal cord injury(SCI).Methods1.Clinical study of Yiqi Huoxue Recipe in the treatment of mild CSM with Qi deficiency and blood stasis syndromeA prospective randomized controlled study was conducted.The study included the patients of mild CSM with Qi deficiency and blood stasis syndrome who met the inclusion and exclusion criteria in the orthopaedic clinic of Guangdong Province Hospital of Traditional Chinese Medicine from December 2020 to November 2021.They were divided into control group and experimental group by random number table method.The control group was treated with mecobalamin tablets,and the experimental group was treated with Yiqi Huoxue Recipe on the basis of the control group.The two groups were treated for 4 weeks.The general data of the two groups were recorded,including gender,age and course of disease.The Japanese Orthopaedic Association(JOA)scores,spinal cord functional status scores(40 points)and the TCM syndrome scores were recorded before treatment,2 weeks after treatment,4 weeks after treatment and 3 months after finishing the treatment.The clinical curative effect was evaluated and the progress of the disease and adverse reactions were observed.2.Experimental study of nanoparticles of the active ingredients from Chinese herbs of supplementing Qi and activating blood circulation for repairing SCI(1)Huangqi(Astragali Radix)and Danshen(Salvia miltiorrhiza Bunge)are the sovereign drugs of Yiqi Huoxue Recipe.Their active ingredients Astragalus Polysaccharidesis(APS)and Tanshinone IIA(TSIIA)were selected for nanocrystallization.Using selenium nanoparticles(SeNPs)as a drug carrier,the SeNPs surface decorated with APS and loaded with TSIIA were synthesized(TSIIA@SeNPs-APS)by redox reaction of selenite with ascorbic acid.The microstructure of TSIIA@SeNPs-APS was characterized by the transmission electron microscope(TEM)and atomic force microscopy(AFM).The particle sizes and Zeta potential were analyzed by a Malvern Zetasizer Nano ZS.The chemical structure was characterized by X-ray photoelectron spectroscopy(XPS),fourier transform infrared(FT-IR)spectroscopy and ultraviolet-visible(UV-Vis)absorption spectrum.(2)Fluorescence intensity of couramin-6-labelled TSIIA@SeNPs-APS in PC 12 cells at multiple time points was assessed by flow cytometry to detect the cellular uptake of TSIIA@SeNPs-APS and the inhibition of different endocytosis inhibitors on the cellular uptake of TSIIA@SeNPs-APS.In addition,lysosomes and nuclei of PC 12 cells were labeled by LysoTracker and DAPI probes and the intracellular trafficking of TSIIA@SeNPs-APS was observed under fluorescence microscope.(3)In this study,tert-butyl hydroperoxide(t-BOOH)was used as an oxidative stress damage induction to PC 12 cells in order to develop an in vitro model of SCI and further studied the repair effects and mechanism of TSIIA@SeNPs-APS in PC 12 cells.CCK-8 method was used to detect the effect of TSIIA@SeNPs-APS on the viability of PC12 cells damaged by t-BOOH,and compared with APS,TSIIA,the SeNPs without APS decoration(SeNPs)and the SeNPs decorated with APS but not loaded with TSIIA(SeNPs-APS).The groups of CCK-8 were as follows:① control group;②t-BOOH group;③t-BOOH+SeNPs group;④t-BOOH+SeNPs-APS group;⑤t-BOOH+TSIIA@SeNPs-APS group;⑥t-BOOH+APS group;⑦t-BOOH+TSIIA group.Next,the cell cycle distribution was analyzed by flow cytometry with propidium iodide(PI)and the apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI double staining and the mitochondrial membrane potential was analyzed by flow cytometry with JC-1 staining.The changes of intracellular total reactive oxygen species(ROS)in PC 12 cells were observed by 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)staining.The expressions of CDK2,pro-caspase-3,pro-caspase-9,Bcl-2,Bcl-xl,Bax and Bad proteins in PC 12 cells were detected by Western blot.(4)Two months old SD female rats were divided into the following groups:① sham group;②SCI group;③APS group;④TSIIA group;⑤SeNPs group;⑥TSIIA@SeNPs-APS group.The above drugs were administered by intraperitoneal injection once a day for 21 consecutive days.The SCI of rat model was established using modified Allen’s method.The recovery of locomotor function of SCI rats was assessed by the Basso-Beattie-Bresnahan(BBB)test and inclined plane test at 1st,3rd,5th,7th,14th and 21st day after operation.The footprint analysis analysis was conducted at 14th day after operation.On the 21st day after operation,the tissues of rats in each group were extracted and sliced for HE staining,Nissl staining and TUNEL+DAPI double staining.The activity of total glutathione peroxidase(GSH-Px)and the content of malondialdehyde(MDA)in the spinal cord tissue were detected.Results1.Clinical study of Yiqi Huoxue Recipe in the treatment of mild CSM with Qi deficiency and blood stasis syndrome83 cases were included,including 41 cases in the control group and 42 cases in the experimental group.There was no significant difference in gender,age,course of disease,and the JOA scores,spinal cord functional status scores and TCM syndrome scores before treatment between the two groups(P>0.05).The JOA scores of the two groups at each time point after treatment were significantly higher than those before treatment(P<0.05).The spinal cord functional status scores of the control group 4 weeks after treatment and 3 months after finishing the treatment were significantly higher than those before treatment(P<0.05).The spinal cord functional status scores of the experimental group at each time point after treatment were significantly higher than those before treatment(P<0.05).The TCM syndrome scores of the two groups at each time point after treatment were significantly lower than those before treatment(P<0.05).Three months after finishing the treatment,the total effective rate of the control group was 80.49%which was significantly lower than 92.86%of the experimental group(P<0.05).There were no cases of aggravation to moderate and severe CSM during follow-up.At the same time,there were no adverse events such as allergy and gastrointestinal reaction.2.Experimental study of nanoparticles of the active ingredients from Chinese herbs of supplementing Qi and activating blood circulation for repairing SCI(1)In this study,TSIIA@SeNPs-APS with uniform particle size,regular morphology and good dispersion were successfully synthesized.The particle size was 80.8 ± 2.5 nm and the zeta potential was-16.6±1.1 mV,and it could exist stably in aqueous solution for at least 4 weeks with good stability.XPS showed that there was an interaction between APS and Se to make the SeNPs more stable.FT-IR and UV-Vis spectra showed that TSIIA@SeNPs-APS was successfully modified by APS and loaded with TSIIA.(2)The results showed the cellular uptake of TSIIA@SeNPs-APS increased in a timeand dose-dependent manner with the highest accumulation in 2 h and maintained the high cellular uptake level within 6 h.TSIIA@SeNPs-APS was taken up by PC 12 cells through clathrin-mediated and caveolae-mediated endocytosis,and then located in the lysosome and released to the cytoplasm.(3)TSIIA@SeNPs-APS effectively improved the viability of PC 12 cells damaged by t-BOOH,and when the concentration was 2.5 μM,the effect of the improvement was the best.Compared with other treatment groups,the difference was statistically significant(P<0.05).PI staining flow cytometry analysis showed that TSIIA@SeNPs-APS reduced the sub-G1 population from 19.2%to 4.4%,the S phase proportion from 19.3%to 9.7%.Annexin V-FITC/PI double staining flow cytometry analysis showed that the proportion of early apoptotic cells from 29.4%to 11.8%.JC-1 staining flow cytometry analysis showed that the percentage of cells with depolarized mitochondria decreased from 78.6%to 40.2%.DCFH-DA staining showed that the total intracellular ROS level decreased from 792.8%to 553.6%.At the same time,Western blot showed that TSIIA@SeNPs-APS significantly reversed the down-regulation of CDK2,pro-Caspase-3,pro-Caspase-9,Bcl-2 and Bcl-xl proteins by t-BOOH and the up-regulation of Bax and bad proteins by t-BOOH.(4)The BBB score(10.83± 1.47)and inclined plate test angle(34.17 ± 5.85°)in TSIIA@SeNPs-APS group were higher than those in SCI group,APS group,TSIIA group and SeNPs group at 3 weeks after operation(P<0.05).In the footprint analysis,rats in the SCI group showed obvious paralysis and extensive drag of hindlimbs unable to support weight and walk normally.Compared with other treatment groups,rats in TSIIA@SeNPs-APS group could better support weight and showed an improvement in coordinated motor functions.HE staining showed that gray structure disorder,obvious necrosis and cavity,irregular neurons and pyknotic nuclei,and a large number of motor neurons lost could be observed in the SCI group.Compared with SCI group,the necrotic cavity of spinal cord tissue in TSIIA@SeNPs-APS group was reduced,and the nucleolus was clearer,and the motor neurons were partially preserved.Nissl staining showed that there were cavities in the spinal cord tissue of SCI group,and the Nissl body was fuzzy,unclear,and shallow.Compared with SCI group,the morphological structure of Nissl body in TSIIA@SeNPs-APS group was improved,indicating that the function of neurons was restored to a certain extent.TUNEL+DAPI double staining showed that there were a large number of TUNEL positive cells in the SCI group.Compared with SCI group,the fluorescence intensity of TUNEL positive cells in TSIIA@SeNPs-APS group decreased significantly.Compared with the SCI group,GSH-Px activity in spinal cord tissue of TSIIA@SeNPs-APS group increased significantly(P<0.001).Compared with the SCI group,MDA content in spinal cord tissue of TSIIA@SeNPs-APS group decreased significantly(P<0.001).Conclusion1.Yiqi Huoxue Recipe has a definite therapeutic effect on patients of mild CSM with Qi deficiency and blood stasis syndrome.It can effectively improve the spinal cord function,slow down the progress of the patient’s condition and improve the syndrome of Qi deficiency and blood stasis.2.The biological activities of APS and TSIIA in the repair of SCI have been improved after nanocrystallization.TSIIA@SeNPs-APS had a protective effect against oxidative stress-induced cytotoxicity in PC 12 cells.The mechanism might be through inhibiting the excessive production of ROS,so as to reduce the damage of mitochondria,reduce cell apoptosis and S phase cell cycle arrest,and finally promote the survival of cells.At the same time,TSIIA@SeNPs-APS could improve the locomotor dysfunction and promote the recovery of locomotor function in SCI rats by reducing the level of oxidative stress in spinal cord tissue to reduce the neuronal damage and apoptosis of anterior horn motor neurons in SCI rats.