Modulatory Effects of Astragalus Polysaccharides on T-Cell Polarization in Mice with Polymicrobial Sepsis

Background. This study evaluated the impact of different doses of Astragalus polysaccharides (APS) on the functional status and phenotype of T cells during polymicrobial sepsis. Methods. On day 1 after cecal ligation and puncture, mice were treated with either saline, 100 (A100), 200 (A200), or 400 mg APS/kg body weight (BW) (A400) by an intraperitoneal injection daily for 4 days. All mice were sacrificed 5 days after the operation. Results. APS treatment reversed the sepsis-induced decrement in the T helper (Th) cell population, and the percentage of activated Th cells also increased in the spleen and Peyer's patches. APS administration downregulated the percentages of circulating Th2 cells and regulatory T cells (Treg), and the percentage of Th17 cells in blood was upregulated in the A400 group. Weight loss and kidney injury were attenuated in the A100 and A200 groups but not in the A400 group at the end of the study. Conclusions. Treatments with 100 and 200 mg APS/kg BW reduced Treg populations and elicited a more-balanced Th1/Th2 response that consequently attenuated immunosuppression in polymicrobial sepsis. High-dose APS administration led to excessive responses of Th17 cells which may have adverse effects in sepsis-induced organ injury.


Procedure for synthesis of Zn 2+ and Cu 2+ complexes [3a-j]
The solution of metal salt [ZnCl 2 , CuCl 2 ] dissolved in ethanol was added gradually to a stirred ethanolic solution of the Schiff base hydrazones [2a-e], in the molar ratio 1:2. The reaction mixture was further stirred for 2-4 h at 60 0 C. Then it was cooled in ice bath to ensure the complete precipitation of the formed complexes. The precipitated solid complexes were filtered and washed four times with water. Finally, the complexes were washed with diethyl ether and dried in vacuum desiccators over anhydrous CaCl 2 .

Elemental Analysis of metal complexes:
The quantitative estimation of Zn 2+ and Cu 2+ has been done by complexometric titration with standard EDTA solution. In a titration an accurately known mass of metal complex is dissolved in an aqueous solution by chemical treatment such as acid-digestion of solid metal complex samples and diluted with high purity water to an accurately known volume. Then an accurately known volume of the aliquot is pipetted into a titration vessel and the analyte of interest is carefully titrated with a standardized EDTA solution to the endpoint of the titration.
2. Place copper complex in porcelain dish and add concentrated 5 mL H 2 SO 4 . Heat on sand bath near dryness. Repeat it for 2 times more.
Then add 5 mL of concentrated HCL and heat again to get Zn (II) in chloride (water soluble) form. Heat this nearly to dryness and extract with deionized water.
3. Carefully dilute sample solution in the 250 mL volumetric flask to the mark with deionized water. Mix it thoroughly. 4. Pipette out 25 mL aliquot into conical flask. Add 15 mL of deionized water, 9-10 mL of pH 10 buffer, and 3 drops of Eriochrome Black T immediately prior to titrating a sample. Titrate with standardized EDTA until the pink solution turns light blue.
5. Calculate the milligrams of zinc in the total sample. Remember that each aliquot represents one tenth of the total sample volume of a 25 mL aliquot titrated out of 250 mL total volume.

CALCULATIONS:
The molarity of the Zn 2+ standard solution (M Zn ) is calculated in normal fashion using the molar mass of Zinc Chloride weighed out and the total volume in liters of the standard solution prepared. And the total mass of Zn in the original 250 mL sample is therefore 10 times this amount.

Estimation of Copper
Experimental Procedure: 1. Weigh accurately three approximately 0.1 g samples of the copper complex.
2. Place copper complex in porcelain dish and add concentrated H 2 SO 4 5mL. Heat on sand bath near dryness. Repeat for 2 times more. Then add 5 mL of concentrated HCL and heat again to get Cu (II) in chloride (water soluble) form. Heat nearly to dryness and extract with deionized water and dilute to 100 mL in standard volumetric sample. Transfer 25 mL in conical flask.
3. Add three drops of indicator to sample, Titrate each sample with the standardized EDTA. The light yellow solution turns green near the end point, then suddenly purplish blue at the end point.
4. Calculate the milligrams of Copper in the total sample. Remember that each aliquot represents one fourth of the total sample volume of a 25 mL aliquot titrated out of 100 mL total volume.

Calculations:
During the titration, the EDTA 2forms a more stable complex and frees the indicator, which then displays its original color. The appearance of the free indicator means that all metal ions have been complexed by EDTA 2-, which signals the end point. At the end point, the following equation applies: N EDTA × V EDTA = N Cu (II) × V Cu (II) = meq Cu(II), V is given in mL The mass of Cu (II) = eq Cu(II)) × (equivalent mass of Cu(II))

SAR Study:
The Cresset's software Forge is a molecular design and SAR (structure activity relationship) interpretation tool that generates and uses molecular alignments as a way to make meaningful comparisons across chemical series. The interaction between a ligand and a protein involves electrostatic fields and surface properties (e.g. hydrogen bonding, hydrophobic surfaces and so on). Two molecules which both bind to a common active site tend to make similar interactions with the protein and hence have highly similar field properties. Accordingly, using these properties to describe molecules is a powerful tool for the medicinal chemist as it concentrates on the aspects of the molecules that are important for biological activity. In Forge, molecules can be aligned by using the Fields of the molecules, by using shape properties or by using a common substructure. Using the Fields gives a 'protein's view' of how the molecules would line up in the active site, generating ideas on how molecules with different structures could interact with the same protein. Using substructure or common shape properties shows how the Fields around a single chemical series varies with activity and in many cases these can be automatically examined to give a 3D quantitative structure active relationship (QSAR) with predictive power for new ideas for synthesis.

Interpretation of Field Point Patterns
Molecules

Antitubercular Studies:
The antitubercular activity of the hydrazone ligands and their metal complexes was tested against Mycobacterium tuberculosis (H37 RV strain) ATCC No-27294 to find out their potency as antimicrobial agent by MIC method (minimum inhibitory concentration).

Microbiological Method:
The antibacterial activity of hydrazones and their metal complexes were tested against Mycobacterium tuberculosis using microplate Alamar Blue assay (MABA). This methodology is non-toxic and reagents used are thermally stable. It also shows good correlation with proportional and BACTEC radiometric method and reproducible results.
To minimize the evaporation of medium in the test wells during incubation 200µl of sterile deionized water was added to all outer perimeter wells of sterile 96 wells plate. The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The concentration of the test sample was prepared between 100 to 0.8 µg/ml ranges. These plates were covered and sealed with paraffin and incubated at 37ºC for five days. Then in next step 25µl of freshly prepared 1:1 mixture of Almar Blue Reagent Tween 10% and 80% was added to the plate and incubated for 24 hrs in incubator. A blue color in the well was indicates bacterial growth whereas pink color show growth of bacteria. From this experiment the MIC can be defined as lowest drug concentration which prevented the color change from blue to pink.