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Cash loans herbal extraction was performed in 160 mL of boiling water for 2 hours, and the herbs were extracted twice. For the second extraction of KXS, the residue from the first extraction was filtered: the same extraction condition was applied on the filtered residue. This extraction condition was also applied to each individual herb as well as a combination of herbs under the cash loans extraction method as described above.

The brain tissues were added with RNAzol reagent (1. The concentration of extracted RNA was calculated from UV absorbance cash loans 260 nm. Quantification of the cDNA was determined by UV absorbance at 260 nm and 280 nm by NanoDrop. The SYBR green signal cash loans detected by Applied Biosystems 7500 Fast Real-Time PCR System.

Real-time PCR products were analyzed by gel electrophoresis on a 1. The reaction was stopped with 1 M sulfuric acid and absorbance recorded at 450 nm immediately. The values of standards and samples were corrected by subtracting the absorbance of nonspecific blinding.

All samples were measured in duplicate in the same assay to minimize interassay variation. Statistical tests were performed with t-test (version 13. Cultured astrocytes reached confluence on the day in vitro (DIV) 12 having the mRNA level of glial fibrillary acidic protein (GFAP), a marker for astrocyte, reaching the peak on DIV 12 to 16 (Figure 1(a)).

To test the culture purity, the astrocytes were seeded onto the glass cover slip. At DIV 12, the culture was fixed for immunofluorescent staining with GFAP and possible contamination of oligodendrocytes, which was viewed under the microscopy (Figure 1(b)).

The result showed that the majority of cell population was astrocyte, and thus, this stage of astrocyte was used subsequently for all biochemical analyses. Meanwhile, cultured astrocytes were stained by Cy3-anti-GFAP (shown in red) and anti-rabbit anti-oligo2 (shown in green) polyclonal antibodies observed under confocal microscopy in the presence of 0.

Representative figures are showed here. Historically, three formulations of KXS have been described, denoted as KXS-652, KXS-984, and DZW-652 (Table 1), and all of them are commonly used today clinically. The notation was described according to the year of their publication. KXS-652 with a ratio of 1 : 1 : 25 : 50 of Ginseng Radix et Rhizoma : Polygalae Radix : Acori Tatarinowii Rhizoma : Poria. Meanwhile, a herbal formula named Ding-Zhi-Wan (DZW-652) was also described with the ratio of 3 : 2 : 2 : 3.

In addition, KXS-984 was recorded in a herbal ratio of 1 : 1 : 1 : 2. The chemical standardizations of these herbal decoctions were done by HPLC fingerprints and quantitation of chemical ingredients (Supplementary Figure 1), as described fully in Zhu et al. The chemical properties of these herbal extracts were prerequisite for all biochemical analyses.

The effect of the three formulations of KXS on the expression of neurotrophic factors was evaluated by quantitative PCR, which included NGF, BDNF, GDNF, NT3, NT4, and NT5.

The specific primers of these neurotrophic factors were listed in Supplementary Table 1. Astrocytes cultured at DIV 12 were treated with KXS from 0. The KXS treatment significantly increased the mRNA levels of NGF, BDNF, GDNF, and NT3 in dose-dependent manners (Figure 2). For BDNF, DZW-652 increased the mRNA expression level over 2. The best effect of DZW-652 could be also observed in GDNF induction.

For NT4 and NT5, there were slight changes observed after KXS treatment (Figure 2). Thus, KXS having a high ratio of Ginseng Radix et Rhizoma and Polygalae Radix could benefit the expression of neurotrophic factors, as this was in DZW-652 formulation. Firstly, standard curves of three target proteins in ELISA assays were made by different concentrations of protein standards.

The coefficient of intra and interassay was over 0. In astrocytes treated with KXS extracts, the induction of NGF, GDNF, and BDNF was determined by ELISA. The amount of NGF of cultured astrocytes was 4. Under the treatment of KXS, the expressions of NGF, GDNF, and BDNF were increased in dose-dependent manners (Figure 3(b)).

In addition, the inducing effect of KXS-984 was slightly better than that of KXS-652 (Figure 3(b)). Here, the application of Bt2-cAMP was used as a control. In addition, the water extracts of four individual herbs were tested for their role in the induction of neurotrophic factors, and the effects were not significant. Here, the water extracts of Ginseng Radix et Rhizoma and Polygalae Radix could slightly stimulate the expression of NGF and GDNF (Figure 4), while the other inductions were very small.

In contrast, a combined herbal mixture (i.

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