Inflammatory Markers Estimation:
  1. Dopamine: Brain injury significantly increased the Dopamine levels in brain tissue of disease control [i.e. TBI (25.91 ± 1.08), EPLT (26.46 ± 0.87) groups as compared to normal controls [i.e. Control (19.95 ± 1.99), Sham (20.29 ± 1.45). The treatment groups showed a significant decrease in the levels of NSE when compared to disease controls. The decrease in the concentration of NSE treatment groups was calculated as [i.e. FH (20.95 ± 1.01), FFA (20.4 ± 0.93), VPA (20.29 ± 1), VFA (20.2 ± 1.3)]. The levels of Dopamine were significantly restored by the treatments applied.
  2. Mitochondrial complex-I: Brain injury damaged the ETC machinery of mitochondria which results in apoptosis of the neurons. Brain levels for Mitochondrial complex-I were markedly found to be significantly increased in injury treated [i.e. TBI (6.77 ± 0.47), EPLT (6.73 ± 0.45) groups as compared to normal controls [i.e. Control (4.33 ± 0.69), Sham (4.46 ± 0.73). The decrease in the concentration of treatment groups was calculated as [i.e. FH (5.40 ± 0.55), FFA (4.99 ± 0.72), VPA (4.78 ± 0.49), VFA (4.8 ± 0.48)] which significantly restored the levels compared to injury groups.
  3. Neuron-Specific Enolase: Brain and serum levels for NSE were markedly found to be significantly increased in injury treated [i.e. brain tissue: TBI (22.33 ± 2.64), EPLT (22.44 ± 3.2) and blood serum: TBI (10.4 ± 0.8) EPLT (10.8 ± 1.2)] groups as compared to normal controls [i.e. brain tissue: Control (14.58 ± 1.04), Sham (15.25 ± 1.37) and blood serum: Control (7.5 ± 1.2), sham (7.7 ± 0.6)]. But the treatment groups were shown a significant decrease in the levels of NSE [i.e. brain tissue: FH (16.8 ± 2.3), FFA (16 ± 2.04), VPA (15.8 ± 2.3), VFA (16.13 ± 1.98), and blood serum: FH (8.7 ± 1.01), FFA (8.3 ± 1.01), VPA (7.8 ± 0.9), VFA (8.2± 1.1)] when compared to disease controls.
  4. Nerve Growth Factor-Beta: Brain levels for Nerve Growth Factor-Beta (NGF-b) were markedly found to be significantly increased in injury treated [i.e. TBI (94.9 ± 6.1), EPLT (96.9 ± 6.8) groups as compared to normal controls [i.e. Control (52.1 ± 5.1), Sham (53.47 ± 6.2). But the decrease in the NGF-b concentration in treatment groups was calculated as significant [i.e. FH (77.58 ± 8.2), FFA (68.56 ± 7.7), VPA (66.1 ± 7.6), VFA (60.43 ± 8.6)] when compared to disease controls.
  5. Ubiquitin Carboxyl-terminal Hydrolase Isozyme L1: Brain and serum levels for UCHL-1 were markedly found to be significantly decreased in injury treated [i.e. brain tissue: TBI (7.58 ± 0.92), EPLT (7.28 ± 1.13) and blood serum: TBI (4 ± 0.78) EPLT (3.96 ± 0.46)] groups as compared to normal controls [i.e. brain tissue: Control (11.92 ± 1.29), Sham (12.18 ± 1.4) and blood serum: Control (6.9 ± 0.71), sham (6.45 ± 0.7)]. The increase in the concentration of UCHL-1 has been found significant in treatment groups [i.e. brain tissue: FH (9.11 ± 0.75), FFA (9.46 ± 1.10) and blood serum: FH (4.69 ± 0.36), FFA (5.19 ± 0.47)]. The concentration values to standard drug therapy VPA, and its combination with FFA [i.e. brain tissue VPA (10.91 ± 0.52), VFA (11.31 ± 1.18) and blood serum VPA (5.61 ± 0.5), VFA (5.79 ± 0.6)] was found more significantly increased when compared to disease controls.
  6. Interleukin-1beta: Brain and serum levels for IL-1β were markedly found to be significantly increased in injury treated [i.e. brain tissue: TBI (29.117 ± 2.34), EPLT (29.517 ± 3.69) and blood serum: TBI (11.23 ± 1.4) EPLT (11.83 ± 1.5)] groups as compared to normal controls [i.e. brain tissue: Control (9.97 ± 1.9), Sham (10.47 ± 1.46) and blood serum: Control (5.72 ± 1.11), sham (5.87 ± 1.36)]. The decrease in the concentration of IL-1β treatment groups [i.e. brain tissue: FH (14.95 ± 3.13), FFA (12.122 ± 3.08) and blood serum: FH (7.4 ± 1.11), FFA (6.6 ± 0.97)] was found less when compared the concentration values to standard drug therapy VPA, and its combination with FFA [i.e. brain tissue VPA (12.017 ± 1.8), VFA (11.27± 1.61) and blood serum VPA (6.33 ± 0.9), VFA (5.98 ± 0.68)]. The overall treatment groups were shown a significant decrease in the levels of IL-1β when compared to disease controls.
  7. Interleukin-10: Blood serum levels for IL-10 were markedly found to be significantly increased in injury treated [i.e. TBI (279.1 ± 4.3), EPLT (289.5 ± 9.6) groups as compared to normal controls [i.e. Control (200.41 ± 9.1), Sham (210.83 ± 11.2)]. The treatment groups were shown a significant decrease in the levels of IL-10 [i.e. FH (232.08 ± 20.4), FFA (226.25 ± 16.1), VPA (221.6 ± 21.7), VFA (221.43 ± 15.9)] when compared to disease controls.
  8. Tumor necrosis factor-alpha: Brain and serum levels for TNF-α were markedly found to be significantly increased in injury treated groups [i.e. brain tissue: TBI (63.6 ± 5.8), EPLT (63.9 ± 4.8) and blood serum: TBI (17.8 ± 1.87) EPLT (18.13 ± 2.6)] as compared to normal controls [i.e. brain tissue: Control (26.2 ± 6), Sham (35.2 ± 6) and blood serum: Control (13.5 ± 3.71), sham (14 ± 3.22)]. The decrease in the concentration of TNF-α treatment groups [i.e. brain tissue: FH (38.13 ± 3.12), FFA (34.91 ± 6) and blood serum: FH (15.81 ± 3.45), FFA (14.94 ± 1.6)] was found less when compared the concentration values to standard drug therapy VPA, and its combination with FFA [i.e. brain tissue VPA (36.22 ± 3.8), VFA (36.1± 4.4) and blood serum VPA (15.43 ± 2.87), VFA (14.66 ± 2.6)]. But the overall treatment groups were shown a significant decrease in the levels of TNF-α when compared to disease controls.
Relative mRNA Expression: The relative mRNA expression of pro-inflammatory/inflammatory genes has been assessed for overall change (Figure-6).
  1. Cyclooxygenase-2: The overall fold change in COX-2 expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham. A significant decrease in the expression of the COX-2 gene has been found in injury treatment groups i.e. FH, FFA, VPA, and VFA. The overall change in expression was similar in all treatment groups.
  2. Heme Oxygenase-2: The overall fold change in HO-2 expression was found to be significantly less in TBI and EPLT groups when compared to Control and Sham. But a significant increase in the expression of the HO-2 gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA.
  3. Nuclear factor (erythroid-derived 2)-like 2: The overall fold change in Nrf-2 expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the Nrf-2 gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA.
  4. Nuclear Factor-ƙβ: The overall fold change in NF-ƙβ expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. And a significant decrease in the expression of the NF-ƙβ gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA.
  5. Interleukin-1β: The overall fold change in IL-1β expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham. A significant decrease in the expression of the IL-1β gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. FH drug was found less effective than FFA, VPA, and VFA in treatment groups while compared to disease controls.
  6. Interleukin-6: The overall fold change in IL-6 expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the IL-6 gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. The overall change in expression was similar in all treatment groups but FFA efficacy was found less compared to FH, VPA, and VFA.
  7. Interleukin-10: The overall fold change in IL-10 expression was found to be significantly increased in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the IL-10 gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. The VPA was found more effective to reduce the change in expression in all treatment groups.
  8. Tumor necrosis factor-alpha: The overall fold change in TNF-α expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the TNF-α gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. The overall change in expression was similar in all treatment groups.
  9. Protein Kinase-B: The overall fold change in Akt expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the Akt gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. The overall change in expression was similar in all treatment groups but combination therapy VFA was less effective than other treatments.
  10. Phosphatase and tensin homolog: The overall fold change in PTEN expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the PTEN gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. The change in expression was high in the combination VFA group in all treatment groups.
  11. Phosphatidylinositol 3-kinase: The overall fold change in PI-3k expression was found to be significantly high in TBI and EPLT groups when compared to Control and Sham groups. A significant decrease in the expression of the PI-3k gene has been found in disease treatment groups i.e. FH, FFA, VPA, and VFA. The overall change in expression was similar in all treatment groups FH was found less effective.
Hematoxylin-Eosin Staining for Neuronal Injury Scoring: It has been found that the morphology of neurons was intact and good in the case of the Control and Sham groups. They exhibited normal size, shape and were arranged compactly with a prominent nucleus i.e. no apoptosis was observed. The disease groups i.e. TBI and EPLT showed diffuse neuronal injury with features such as rarefaction of neuropil, eosinophilic cytoplasm, shrunken and pyknotic nuclei in hippocampus and cortex region i.e. moderate and severe apoptosis was observed. In the case of treatment groups, we have seen fewer apoptotic neurons as compared to disease controls. The scoring system revealed mild apoptosis in the case of FH, FFA, and VFA but no proper distinguished apoptotic neurons were found in the VPA group (Figure-7).
Along with it, no significant histopathological changes have been found in vital organs i.e. liver and kidneys of injury controls when compared to treatment groups.