Natural product HTP screening for attenuation of cytokine-induced neutrophil chemo attractants (CINCs) and NO2- in LPS/IFNγ activated glioma cells

Abstract

Chronic and acute central nervous system (CNS) inflammation are contributors toward neurological injury associated with head trauma, stroke, infection, Parkinsons or Alzheimers disease. CNS inflammatory illnesses can also contribute toward risk of developing glioblastoma multiforme (GBM). With growing public interest in complementary and alternative medicines (CAMs), we conduct a high throughput (HTP) screening of >1400 natural herbs, plants and over the counter (OTC) products for anti-inflammatory effects on lipopolysaccharide (LPS)/interferon gamma (IFNγ) activated C6 glioma cells. Validation studies were performed showing a pro-inflammatory profile of [LPS 3 µg/ml/ IFNγ 3 ng/ml] consistent with greater release [>8.5 fold] of MCP-1, NO2-, cytokine-induced neutrophil chemo-attractants (CINC) 1, CINC 2a and CINC3. The data show no changes to the following, IL-13, TNF-a, fracktaline, leptin, LIX, GM-CSF, ICAM1, L-Selectin, activin A, agrin, IL-1α, MIP-3a, B72/CD86, NGF, IL-1b, MMP-8, IL-1 R6, PDGF-AA, IL-2, IL-4, prolactin R, RAGE, IL-6, Thymus Chemokine-1, CNTF,IL-10 or TIMP-1. A HTP screening was conducted, where we employ an in vitro efficacy index (iEI) defined as the ratio of toxicity (LC₅₀)/anti-inflammatory potency (IC₅₀). The iEI was precautionary to ensure biological effects were occurring in fully viable cells (ratio > 3.8) independent of toxicity. Using NO2- as a guideline molecule, the data show that 1.77% (25 of 1410 tested) had anti-inflammatory effects with iEI ratios >3.8 and IC₅₀s <250µg/ml. These include reference drugs (hydrocortisone, dexamethasone N6-(1-iminoethyl)-l-lysine and NSAIDS: diclofenac, tolfenamic acid), a histone deacetylase inhibitor (apicidin) and the following natural products; Ashwaganda (Withania somnifera), Elecampagne Root (Inula helenium), Feverfew (Tanacetum parthenium), Green Tea (Camellia sinensis), Turmeric Root (Curcuma longa) Ganthoda (Valeriana wallichii), Tansy (Tanacetum vulgare), Maddar Root (Rubia tinctoria), Red Sandle wood (Pterocarpus santalinus), Bay Leaf (Laurus nobilis, Lauraceae), quercetin, cardamonin, fisetin, EGCG, biochanin A, galangin, apigenin and curcumin. The herb with the largest iEI was Ashwaganda where the IC₅₀/LC₅₀ was 11.1/>1750.0μg/ml, and the compound with the greatest iEI was quercetin where the IC₅₀/LC₅₀ was 10.0/>363.6μg/ml. These substances also downregulate the production of iNOS expression and attenuate CINC-3 release. In summary, this HTP screening provides guideline information about the efficacy of natural products that could prevent inflammatory processes associated with neurodegenerative disease and aggressive glioma tumor growth.

Keywords: Astrocytes; C6 glioma; HTP; Herbs; High throughput screening; Natural products.

Figure 1A

Cytokine release in LPS/IFNγ treated C6 glioma cells at 24h. The blot image (Top) and corresponding array grid layout (Bottom) are presented. (B) CINC1,2a and 3 release were significantly upregulated in LPS/IFNγ treated C6 glioma. The data represents relative density and are expressed as the Mean ± S. E. M., n=4. Differences between resting and activated cells were determined using a Student’s t-test, (*) P<.001.

Figure 1A

Cytokine release in LPS/IFNγ treated C6 glioma cells at 24h. The blot image (Top) and corresponding array grid layout (Bottom) are presented. (B) CINC1,2a and 3 release were significantly upregulated in LPS/IFNγ treated C6 glioma. The data represents relative density and are expressed as the Mean ± S. E. M., n=4. Differences between resting and activated cells were determined using a Student’s t-test, (*) P<.001.

Figure 2A

NO⁻₂ production in resting and LPS/IFNγ treated C6 glioma cells ± selective iNOS inhibitor: L-NIL (20μg/ml) or hydrocortisone (20μg/ml). The data represent NO2− produced (μM) and are expressed as the Mean ± S. E. M., n=4. Differences between resting and activated cells were determined by a Student’s t-test (*) P<.001, and LPS vs. L-NIL and hydrocortisone treated cells [*].

Figure 2B

Cell viability in resting and LPS/IFNγ treated C6 glioma cells ± selective iNOS inhibitor: L-NIL (20μg/ml) and hydrocortisone (20μg/ml). The data represent cell viability as % control and are expressed as the Mean ± S. E. M., n=4. Differences between resting and activated cells were determined by a Student’s t-test (*) P<.001, and LPS vs. L-NIL and hydrocortisone treated cells [*].

Figure 2C

ICC imaging of iNOS expression using rabbit anti mouse iNOS/goat anti-rabbit Alexafluor 488, in fixed permeabilized: Controls: resting C6 cells, LPS/IFNγ treated, LPS/IFNγ treated + L-NIL (20μg/ml) and LPS/IFNγ + hydrocortisone (20μg/ml).

Figure 2D

ELISA quantification of CINC-3 released in supernatant by LPS/IFNγ treated C6 glioma cells at 24 hours ± selective iNOS inhibitor: L-NIL (20μg/ml) and hydrocortisone (20μg/ml). The data represent CINC-3 (pg/μl) and expressed as the Mean ± S. E. M., n=3. Differences between resting and LPS/IFNγ activated cells were determined by a Student’s t-test (*) P<.001, and LPS/IFNγ vs. L-NIL and hydrocortisone treated cells (*) P<.001.

Figure 3

[A] The basic study layout consisted of a primary Tier 1 elimination by which all herbs were tested to reduce LPS/IFNγ induced NO-2 in C6 cells, with maximum working concentrations : 230μg/mL (herbal extracts) and <92μg/ml (metabolites, drugs and polyphenolics). [B] Compounds displaying an IC₅₀ below 1st Tier concentrations were further evaluated where toxicity/anti-inflammatory effects were evaluated simultaneously and an in vitro efficacy index iEI differential is established (LC₅₀/IC₅₀).

Figure 4

Table 1 scatter – plot showing HTP Screening Results by LC₅₀ (μg/ml) X- Axis and IC₅₀ (μg/ml) Y-Axis.

Figure 5

Sample linear regression profiles for dose response used to calculate LC_50s (μg/ml) and IC_50s (μg/ml) where sub lethal-anti-inflammatory single concentrations were used to evaluate for CINC-3 release in LPS/IFNγ glioma demarcated by a (◘). The data represents cell viability and NO2− (% LPS/IFNγ Control), and significance from controls were evaluated with a one-way ANOVA, with a Tukey Post Hoc test, (*) P<.05. The data represents CINC-3 (as % LPS/IFNγ) and expressed as the Mean ± S.E.M., n=3. Differences determined by a Student’s t-test (*) P<.05.

Figure 5

Sample linear regression profiles for dose response used to calculate LC_50s (μg/ml) and IC_50s (μg/ml) where sub lethal-anti-inflammatory single concentrations were used to evaluate for CINC-3 release in LPS/IFNγ glioma demarcated by a (◘). The data represents cell viability and NO2− (% LPS/IFNγ Control), and significance from controls were evaluated with a one-way ANOVA, with a Tukey Post Hoc test, (*) P<.05. The data represents CINC-3 (as % LPS/IFNγ) and expressed as the Mean ± S.E.M., n=3. Differences determined by a Student’s t-test (*) P<.05.