| Objective:In this experiment,the model rats of Parkinson’s disease(PD)made by subcutaneous injection of rotenone into the back of neck were taken as the research object.The effects of moxibustion at GV16(Fengfu),CV4(Guanyuan),ST36(Zusanli)points on PINK1/Parkin pathway in substantia nigra of PD model rats were observed according to the guiding ideology of basic theory of traditional Chinese medicine and modern medical pathological theory.Taking this as an opportunity,the possible mechanism of the prevention and treatment of PD by moxibustion was discussed,and more experimental basis was provided for clinical prevention and treatment of PD by moxibustion.Methods:Forty SPF grade healthy male SD rats(weighing 200±20g)were placed in the barrier system of the animal room for adaptive feeding for two weeks.Two weeks later,16 rats were randomly selected according to the random number table and randomly divided into the normal group(8 rats)and the sham operation group(8 rats).The remaining 24 rats belonged to the model group.1 g rotenone was fully mashed with glass rod and dissolved in the mixed solution of dimethyl sulfoxide(DMSO)and 0.9% NaCl(the volume ratio of DMSO to 0.9% NaCl is 7:1).The final solubility of the mixed solution containing rotenone was 1 mg/mL after constant volume.Theprepared solution was sealed and preserved in brown bottle at low temperature.Rats in the model group were subcutaneously injected with rotenone solution at the back of neck(1 mg/Kg),once a day for 40 days.The sham operation group was received subcutaneous injection of the same dosage of rotenone-free DMSO and 0.9% NaCl solution once a day for 40 days.The normal group was fed normally without any treatment.Forty days after the establishment of the model,14 rats with behavioral score greater than 2 and less than 8 were selected according to Chen Xin’s behavioral scoring criteria.On the second day,two rats were randomly selected from the normal group,the sham operation group and the model group for perfusion.The expression of tyrosine hydroxylase(TH)and α-synuclein(α-syn)in substantia nigra of rats was detected by immunohistochemistry to further determine whether the model was successful.After the identification of behavioral scoring criteria and TH and α-syn in substantia nigra of rats by immunohistochemistry,the remaining 12 rats in the model group were randomly divided into PD model group and the moxibustion treatment group.In the intervention stage of the normal group,the sham operation group and PD model group did not add intervention factors,grabbed with the moxibustion treatment group at the same time every day,fixed in rat clothes.In the moxibustion treatment group,GV16(Fengfu),CV4(Guanyuan),ST36(Zusanli)acupoints were selected,and 7 mm moxibustion bars were used at the same time every day,once a day,15 minutes for each acupoint,7 days for a course of treatment,a total of 2courses of treatment.After the treatment,the rats in each group were scored again,and the rats were sampled the next day.After moxibustion treatment,the expression of PINK1 and Parkin in substantia nigra of rats was detected by Western blotting,and the relative expression of PINK1 and Parkin in substantia nigra of rats was detected by RT-PCR.Results:(1)At the stage of modeling,the behavioral scores of rats inthe model group were significantly higher than those in the normal group and the sham operation group(P<0.01);at the stage of moxibustion treatment,the behavioral scores of rats in the moxibustion treatment group were significantly lower than those in the PD model group(P<0.01),and no significant changes in the behavioral scores were observed in the normal group and the sham operation group.(2)Immunohistochemical staining showed that TH staining was significantly lost and unevenly distributed in the model group compared with the normal group and the sham operation group.Compared with the normal group and the sham operation group,α-syn staining was more abundant and dense in the model group.(3)Western blotting showed that the expression of PINK1 and Parkin in substantia nigra of rats in PD model group was significantly lower than that in the normal group and the sham operation group(P<0.05);compared with the PD model group,the expression of PINK1 and Parkin in substantia nigra of rats in the moxibustion treatment group increased significantly(P<0.05).(4)RT-PCR showed that the relative expressions of PINK1 mRNA 、 ParkinmRNA in substantia nigra of rats in the PD model group were significantly lower than those in the normal group and the sham operation group(P<0.05);compared with PD model group,the relative expressions of PINK1 mRNA 、ParkinmRNA in substantia nigra of rats in the moxibustion treatment group were significantly higher(P<0.05).Conclusions :(1)This experimental study re-validated that rotenone subcutaneously injected into the neck and back can effectively replicate the rat model of PD.(2)Pathological changes of PD were observed in the rat model of rotenone-induced PD,TH expression in substantia nigra was significantly decreased and α-syn expression was significantly increased.(3)Moxibustion can significantly improve the behavior and appearance of PD rats.(4)The expression of PINK1 and Parkin protein in substantia nigra of PD ratsincreased significantly under moxibustion treatment,and the relative expression of PINK1mRNA、ParkinmRNA increased significantly.This may be due to the increased level of mitochondrial autophagy,enhanced ability to degrade related pathological protein and reduced pathological protein aggregation,which may be one of the mechanisms of Moxibustion in the treatment of Parkinson’s disease. |
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