Benificial effect of stachydrine on the traumatic brain injury induced neurodegeneration by attenuating the expressions of Akt/mTOR/PI3K and TLR4/NFκ-B pathway

Animals

Albino rat (Age: 3 weeks; Weight: 17-23g) was used in the given study. All the animals were procured from Taizhou University Medical School, China. Standard guidelines (Humidity: 60 ± 5 %; Temperature: 24 ±3°C) were used to store the animals for 12hr light and dark cycle. Protocols of the investigation were approved by ethics committee of The First Affiliated Hospital of Nanchang University, China (IACUC/FAHNU/2017/08) and the given study followed the guidelines of Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) for experimentation and animal use.

Chemicals

Stachydrine was purchased from MedChem Express, USA and isoflurane was procured from Abbott Laboratories, USA. t-PI3K, p-PI3K, t-Akt, p-Akt, p-mTOR, t-mTOR, Bcl-2, Bax, LC-3, TLR-4, NF-kB and β-actin was procured from Abcam, California, USA. ELISA kits were purchased from Ebioscience Company, USA.

Experimental

Animals were anesthetized before the surgery by administering the pentobarbital 60 mg/kg i.p. and skull of animals were exposed by making an incision to medline. Later a disc of steel having the dimension 3 mm thickness and 10 mm diameter placed at biparietal and coronal suture. Rats were placed on the cushion and a copper hammer of 450 g was fall from the height of 1.5 m. Thereafter skull was cleaned properly and sutured the same.

All the animals were divided in to five different groups such as Normal group which is shame operated group receives only saline solution; TBI group which receives saline solution; stachydrine 30 and 60 mg/kg group which receives stachydrine 30 and 60 mg/kg, i.p. 2 hr after the TBI for the duration of one week.

Estimation of neurological function

Modified Neurological Severity Score (mNSS) was used to determine balance, touch, vision, abnormal behavior, muscle mass, sensation and motion. mNSS was calculated on the 0-18 scale, as 0 shows the normal brain function and 18 shows the sever defect in the neurological function. Neurological functions were determined from day 1, 3, 7 and 14.

Determination of water content in the brain All the animals were sacrificed and the brain was isolated from the rats and analytical balance was used to determine the wet weight of brain and later dry weight of brain tissue was observed by drying it in oven at 100°C for one day.

% water content brain = (wet weight - dry weight)/wet weight × 100%.

Determination of behavioral changes

Morris water maze apparatus was used to determine the behavioral changes. Water maze having height and diameter of 0.5 and 1.2 m respectively and the platform was depth at 15.5 cm. Four quadrants were created in the apparatus with thread and in the one of the quadrant platform was placed in such a way that it was not able to visualized. Observation of swimming behavior was done for the duration of six days continuously and escape latency was determined. Later by escaping the platform spatial memory of each rat was determined and effect of stachydrine was also estimated in the TBI injured rats.

Nissl staining

Isolated brain tissues were fixed by using 4% formalin and further tissues were embedded liquid paraffin. Microtome was used to section the brain tissues of 4 μm thickness and xylene was used to de-paraffinized the tissue. These tissue sections were treated with alcohol to rehydrate the tissues. Nissl staining was used to stain the brain tissues for 5 min. Section of tissues were observed by using trinocular microscope and evaluated randomly by pathologist.

Western blot assay

Brain tissues of injured location were separated out and tissue lysis was done using lysis buffer. Supernatant from the lysed tissue was separated out by centrifuging the lysate for the period of 5 min at 10000 RPM. Later sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the total protein and then separated protein is filtered on nitrocellulose membrane. Further membrane was incubated with I^ry antibodies like t-PI3K (Dilution at a ratio of 1:500), p-PI3K (Dilution at a ratio of 1:500), t-Akt (Dilution at a ratio of 1:500), p-Akt (Dilution at a ratio of 1:500), p-mTOR (Dilution at a ratio of 1:500), t-mTOR (Dilution at a ratio of 1:500), LC-3 (Dilution at a ratio of 1:500), TLR-4 (Dilution at a ratio of 1:1000), NF-ƙB (Dilution at a ratio of 1:1500) and β-actin (Dilution at

a ratio of 1:1000) for overnight at 4°C. Later horseradish peroxidase-conjugated secondary antibody was used to incubate for the period of 60 min at room temperature with total protein. Image J software was used to estimate the band density.

Determination of mediators of inflammation

ELISA kits were used to determine the concentration of mediators of inflammation such as INF- γ, IL-1β and TNF-α in the blood of TBI injured rats.

Statistical Analysis

All data were expressed as mean ± SEM (n = 10). The statistical analysis was performed using one way ANOVA. Post-hoc comparison of means was carried out by Dunnett’s post hoc test (Gradpad prism 6.1., CA, USA) multiple comparisons. The level of statistical significance was set at P < 0.05.

Stachydrine ameliorates the cognitive dysfunction

Assessment of memory impairment in stachydrine treated group in TBI injured rats was shown in Fig.2. Percentage number of crossing and time spent in the target quadrant was found to be less and escape latency was more in TBI group than normal group. Off note, stachydrine reported to improve the memory as percentage number of crossing in TBI injured rats. Time spent in the target quadrant was enhanced and escape latency was reduced in stachydrine group than normal group.

Fig. 2

Stachydrine ameliorate the cognitive dysfunction in TBI injured rats by MWM Mean ± SEM (n = 10), ** p < 0.01 than normal group; ^## p < 0.01 than TBI group

Stachydrine ameliorate the neuronal apoptosis

Nissl staining was done on the cortical tissues to determine the number of apoptotic neurons (Fig.3.). It was seen that the percentage of apoptotice neurons was found more in TBI group (81.6%) than normal group (5.3 %). However stachydrine treatment reduces the percentage of apoptotice neurons upto 32.4 % in TBI injured rats.

Fig. 3

Stachydrine ameliorate the neuronal apoptosis in the cortical tissues of TBI injured rats by Nissl staining Mean ± SEM (n = 10), ** p < 0.01 than normal group; ^## p < 0.01 than TBI group

Stachydrine effect on PI3K/ mTOR/ Akt signaling pathway

Rats treated with stachydrine assessed for the expressions of PI3K/mTOR/Akt proteins by western blot assay (Fig.4.). Expression of p-mTOR/t-mTOR, p-Akt/t-Akt and p-PI3K/t-PI3K was decreased significantly (p<0.01) in the brain tissues of TBI group than normal group. There was reduction in the expression of p-mTOR/t-mTOR, p-Akt/t-Akt and p-PI3K/t-PI3K in brain tissues of stachydrine treated group than TBI group.

Fig. 4

Stachydrine ameliorate the altered expressions of PI3K/mTOR/Akt proteins in the cortical tissues of TBI injured rats Mean ± SEM (n = 10), ** p < 0.01 than normal group; ^## p < 0.01 than TBI group

Stachydrine effect on LC-3, TLR-4 and NF-ƙB

Stachydrine ameliorate the altered expressions of NF-κB, TLR-4 and LC-3 proteins in the brain tissues of TBI injured rats was shown in Fig. 5. There was enhancement of expression of LC-3, TLR-4 and NF-ƙB protein in the tissue homogenate in the TBI rats than normal group. However treatment with stachydrine lowers the expression of NF-κB, TLR-4 and LC-3 proteins in tissue homogenate than TBI group.

Fig. 5

Stachydrine ameliorate the altered expressions of NF-κB, TLR-4 and LC-3 proteins in the brain tissues of TBI injured rats Mean ± SEM (n = 10), ** p < 0.01 than normal group; ^## p < 0.01 than TBI group

Fig. 6

Stachydrine ameliorate the altered level of inflammatory mediators in the blood of TBI injured rats Mean ± SEM (n = 10), ** p < 0.01 than normal group; ^## p < 0.01 than TBI group