Part Ⅱ：Mechanism Of Autophagy Regulation in MPTP-Induced PD Mice Via The MTOR Signaling Pathway By Echinacoside

Contact: emily.li@wecistanche.com

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Improvement of the Neurobehavioral Symptoms in MPTP-Induced PD Mice by Echinacoside

Echinacoside Shortened the Climbing Time in MPTP-Induced PD Mice

The T-turn and T-Total in the M group were noticeably longer than those in the N group (P < 0.01). The T-turn and T-Total in the EH group significantly decreased compared with those in the M group (P < 0.01). Compared with the EH group and M group, the above two indexes in the CQ group and WO group increased significantly (P < 0.01 and P < 0.05, respectively). Comparisons between the EH group and RA group showed there was no significant difference in T-turn and T-Total (as illustrated in Figure 1A).

Echinacoside is good for health

Echinacoside Increased the Spontaneous Activity in MPTP-Induced PD Mice

The rearing number within five minutes, and the total distance within 30 minutes all significantly decreased in the M group compared with those in the N group (P < 0.01). The above two indexes significantly increased in the EH group and the RA group compared with those in the M group (P < 0.01). Compared with the EH group, the rearing number and the total distance significantly decreased in the CQ group and the WO group (P < 0.01). There were no statistically significant differences in the above two indexes between the EH group and RA group (P > 0.05) (as shown in Figure 1B).

Figure I The neurobehavior in mice of each group.

（A） The pole test in mice of each group∶ Turning time （T-Tumn）; Total dimping time （下Totl）.

（B） The spontaneous activity in mice of each group: The erection times within five minutes (Rearing number); The horizontal movement distance within 30 minutes (Total Distance).

(C)The gait analysis in mice of each group: The stride length of forelimb; The stride length of the hind Limb.

(D)The rotarod in mice of each group: The staying time on the rotarod (also known as the Latency to fall）

Notes:*P<0.05,**P <0.0I, compared with the model group;#*p<0.0l, compared with the high-dose echinacoside group, n =6.

Echinacoside Prolonged the Fore and Hind Limb Strides in MPTP-Induced PD Mice

The fore and hind limb stride significantly reduced in the M group compared with those in the N group (P < 0.01). The fore and hind limb stride significantly increased in the EH group and RA group compared with those in the M group (P < 0.01). Compared with the EH group and the M group, the fore and hind limb strides were significantly reduced in the CQ group and the WO group (P < 0.01 and P < 0.05, respectively). There were no statistically significant differences in fore and hind limb strides between the EH group and RA group (as demonstrated in Figure 1C).

Echinacoside Prolonged the Latency to Fall in MPTP-Induced PD Mice

The staying time on the rotarod, also known as the latency to fall, was significantly reduced in the M group compared with the N group (P < 0.01). The latency to fall significantly increased in the EH group and the RA group compared with the M group (P < 0.01). The latency to fall significantly reduced in the CQ group and WO group (P < 0.01 and P < 0.05, respectively) compared with the EH group and M group. There were no statistically significant differences in the latency to fall between the EH group and RA group (P > 0.05) (as shown in Figure 1D).

Echinacoside is good for health

Echinacoside Increased the Content of DA and Metabolites in the Striatum in MPTP-Induced PD Mice

Compared with the N group, the contents of DA, DOPAC, and HVA in the M group significantly reduced (P < 0.01). The contents of DA, DOPAC and HVA significantly increased in the EH group and the RA group compared with those in the M group (P < 0.01). Compared with the EH group and M group, the contents of DA, DOPAC, and HVA significantly decreased in the CQ group and the WO group (P < 0.01 and P < 0.05, respectively). There were no statistically significant differences in the contents of DA, DOPAC, and HVA between the EH group and the RA group (P > 0.05) (as shown in Figure 2A–C). Compared with the N group, the content of 5-HT significantly decreased in the M group (P < 0.01), and the content of 5-HT in the EH group significantly increased (P < 0.01). Compared with the EH group, the content of 5-HT significantly decreased in the CQ group and the WO group (P< 0.01) (as demonstrated in Figure 2D).

Figure 2 Detection of the neurotransmitter contents in the striatum by the High-performance liquid phase electrochemical method (HPLC-ECD).

(A)Dopamine (DA):

(B)dihydroxsyphenybacetic acid (DOPAC);

(C) homovanllic acid (HVA);

(D) 5-hydroxytryptamine (5-HT).

Notes:*P < 0.05.*P<00 I. compared with the model group; "P<0.05."P<001.compared with the high-dose echinacoside group

Echinacoside Increased the Expression of TH in the Substantia Nigra and Decreased the Expression of α-Synuclein in PD Mice

First, double immunofluorescence staining was conducted for TH and α-synuclein. Compared with the N group, the expression of TH in the substantia nigra significantly reduced in the M group, while the expression of α-synuclein significantly increased. However, compared with the M group, the expression of α- synuclein significantly decreased in the EH group and the RA group. The expressions of α-synuclein significantly increased in the CQ group and the WO group, which were even higher than that in the M group (as demonstrated in Figure 3).

Figure 3 immunofluorescence double staining of TH and a-synuclein in the substantial nigra in mice of each group.

The figure showed the immunofluorescence double staining of tyrosine hydroxylase (TH)

α-synuclein in the substantia nigra in mice of each group n =6

the magnification of the fluorescence microscope: 200×.TH was bleed with green fluorescence,

2nd a-student was bleeding with red fluorescence, the blue DAPI represented the nudes.

The overlapping of green 2nd red was the double-stained positive cell (yellow).

Then, a Western blot was adopted to measure the above two indicators. The results showed that the protein expression of TH in the M group was significantly lower than that in the N group (P < 0.01). The protein expressions of TH in the EH group and RA group significantly increased compared with that in the M group (P < 0.01). The protein expressions of TH in the CQ group and WO group were significantly decreased compared with that in the EH group (P < 0.01). The protein expression of α-synuclein in the M group also increased compared with the N group (P < 0.01). However, the protein expressions of α- synuclein in the EH group and the RA group significantly decreased compared with that in the M group, and the differences were statistically significant (P < 0.01). Compared with the EH group and the M group, the protein expressions of α-synuclein in the CQ group and the WO group increased significantly, and the differences were statistically significant (P < 0.01) (as shown in Figure 4).

Figure 4 The changes of protein expressions of TH and α-synuclein in the substantial nigra in mice of each group.

Notes∶甲P<0.05，**p<00I，compared with the model group; "P<005，"P<00I，compared with the high-dose echinacoside group n=6.

Echinacoside Decreased the Expression of P62 in the Substantia Nigra in MPTP-Induced PD Mice

First, a Western blot was adopted to detect the protein expression of P62. The results revealed that the protein expression of P62 in the M group increased noticeably compared with the N group (P < 0.01). The protein expressions of P62 in the EH group and RA group decreased noticeably compared with the M group, and the differences were statistically significant (P < 0.01). Compared with the EH group, the protein expressions of P62 in the CQ group and WO group increased noticeably (P < 0.01) (as shown in Figure 5C). Then, immunofluorescence staining was conducted for P62 and DAPI. It was found that compared with the N group, the P62 in the substantia nigra increased more obviously in the M group, the expressions of P62 decreased in the EH group and the RA group, and expressions of P62 increased in the CQ group and the WO group (as demonstrated in Figure 5D).

Figure 5 The changes of protein expressions of Becin I, LC3ll P62, and P62 immunofluorescence staining in the substantia nigra in mice of each group.

Notes:*P< 005,*P<00l, compared with the model group;"P< 0.05,""P< 0.0I, compared with the high-dose echinacoside group

The magnification of the fluorescence microscope: 200×.P62 was Labeled with red fluorescence. The blue DAPI represented the nude, n= 6.

Echinacoside Increased the Expression of Beclin 1 and LC3II in the Substantia Nigra in MPTP-Induced PD Mice

First, a Western blot was adopted to determine the protein expression of Beclin 1 in the substantia nigra in mice of each group. It was found that the protein expression of Beclin 1 in the M group increased significantly compared with the N group (P < 0.01). However, the increases in protein expression of Beclin 1 in the EH group and the RA group were more obvious (P < 0.01). Compared with the EH group and the M group, the protein expressions of Beclin 1 in the CQ group and the WO group decreased significantly, and the differences were statistically significant (P < 0.01) (as demonstrated in Figure 5A).

Because LC3 is a key indicator of autophagy, double staining was conducted for TH and LC3. The results showed that the number of LC3 positive cells in the M group significantly increased compared with that in the N group. The number of LC3 positive cells in the EH group and the RA group increased more significantly than in the M group. However, when using the autophagy inhibitor, the number of LC3 positive cells in the WO group was significantly lower than that in the M group, while the number of LC3 positive cells did not decrease significantly in the CQ group (as shown in Figure 6). Then, a Western blot was used to detect the protein expression of LC3 in the substantia nigra in mice of each group. The results indicated that the protein expression of LC3II in the M group significantly increased compared with the N group (P < 0.01). Protein expressions of LC3II in the EH group, RA group, and CQ group all increased significantly compared with the M group (P < 0.01). In the WO group, the protein expression of LC3II decreased compared with the EH group and the M group, and the differences were statistically significant (P < 0.01 and P < 0.05, respectively) (as demonstrated in Figure 5B).

Echinacoside is good for health

The Changes of the Substantia Nigra Ultrastructure and Autophagosomes Observed by Transmission Electron Microscope

Through the observation of the substantia nigra ultrastructure, it was found that, except for the N group, the mice all showed varying degrees of substantia nigra ultrastructure injury, manifested as swelling of mitochondria, expansion of rough endoplasmic reticulum, expansion of Golgi, increase of lysosomes, and forming of autophagosomes with double membrane structure. The number of autophagosomes in the EH group, RA group, and CQ group significantly increased compared with other groups. The number of autophagosomes in the CQ group was higher than that in the M group, which was consistent with the results of an increase in the protein expression of LC3II in the fluorescence double staining and Western blot. It also suggested that the number of LC3II and autophagosomes were correlated to a certain extent. There was no obvious increase in autophagosomes in the WO group, suggesting that in this group, the increase of autophagy caused by echinacoside was inhibited (as demonstrated in Figure 7).

Figure 7 The ultrastructural changes and formation of autophagosome in the substantial nigra in mice of each group.

(A)The normal saline control group, with the normal structure of the substantia nigra;

(B)the model group, with the manifestations of mitochondria swelling, rough endoplasmic reticulum expansion, and formation of lysosome and autophagosome:

(C and D) the echinacoside work. with relatively more formation of lysosome and autophagosome:

(E) the rapamycin group, with manifestations of mitochondria swelling and formation of lysosome and autophagosome;

(F)the chloroquine group, with manifestations of mitochondria swelling and formation of lysosome and autophagosome:

(G and H) the wortmannin group. with noticeably swollen mitochondria, expanded rouch edopbs mik reticulum

(G), and swelling of mitochondri2. expansion of rough endoplasmic reticulum, obvious expansion df the Gds and increase of Mysosomes.

The Effect of Echinacoside on Protein Expression of AKT and mTOR in Substantia Nigra in MPTP-Induced PD Mice

Western blot was used to detect the protein expression of AKT and mTOR in each group. The results revealed that compared with the N group, the protein expression of p-AKT/AKT decreased noticeably in the M group (P < 0.01). The protein expressions of p-AKT/AKT in the EH group and RA group increased noticeably compared with the M group (P < 0.01), and the protein expression of p-AKT/AKT in the EH group was higher than that in the RA group (P < 0.01). Compared with the EH group, the protein expressions of p-AKT/AKT in the CQ group and WO group decreased noticeably (P < 0.01) (as shown in Figure 8). Also, it was found that the protein expression of p-mTOR/mTOR in the M group was slightly lower than that in the N group (P < 0.05), but the decrease in protein expression of p-mTOR/mTOR was more significant in the EH group and the RA group than in the M group (P < 0.01). Compared with the EH group, the protein expression of p-mTOR/mTOR increased in the CQ group and the WO group (P < 0.01) (as demonstrated in Figure 8, the lower part).

Figure 8 The changes of protein expressions of AKT and mTOR in the substantial nigra in mice of each group.

Notes: *P < 0.05, **P < 0.01, compared with the model group; ##P< 0.01, compared with the high-dose echinacoside group, n= 6.

Echinacoside is good for health

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