Acacia ferruginea inhibits cyclophosphamide-induced immunosuppression and urotoxicity by modulating cytokines in mice

Figures & data

Figure 1. Effect of treatments on blood parameters in mice at various timepoints during the treatment/post-treatment period. (A) Total white blood cell [WBC] count. (B) Hemoglobin [Hb] content. Mice were given a total of 10 daily doses of CTX; every 3 days - starting prior to the first treatment (Day 0) and during/after the treatment (up to 20 days after final treatment) - blood was collected from the tail vein. From this material, total WBC counts and Hb levels were measured. Values shown are mean ± SD. ^ap < 0.01, ^bp < 0.05 for CTX only vs CTX/extract- or CTX/MESNA-co-treated. ^cp < 0.01, ^dp < 0.05 for normal versus A. ferruginea extract alone.

Figure 2. Effect of treatments on body weight (BW) of mice at various timepoints during the treatment/post-treatment period. Every 3 days - starting prior to the first treatment (Day 0) and during/after the treatment (up to 20 days after the final treatment, i.e. Day 30) - the BW of each animal was measured. ^ap < 0.01, ^bp < 0.05 for CTX only versus CTX/extract- or CTX/MESNA-co-treated.

Table 1. Effect of treatments on organ indices on Days 7 and 11.

	Relative organ weights (g/100 g BW)
	Spleen		Thymus		Kidney		Liver		Urinary Bladder
Treatment	Day 7	Day 11	Day 7	Day 11	Day 7	Day 11	Day 7	Day 11	Day 7	Day 11
Normal	0.39 ± 0.01	0.42 ± 0.03	0.21 ± 0.02	0.22 ± 0.01	1.10 ± 0.09	1.14 ± 0.07	3.21 ± 0.12	3.24 ± 0.10	0.07 ± 0.02	0.08 ± 0.02
CTX only	0.32 ± 0.03	0.34 ± 0.02	0.18 ± 0.05	0.19 ± 0.04	1.41 ± 0.16	1.43 ± 0.23	3.39 ± 0.09	3.42 ± 0.13	0.12 ± 0.02	0.11 ± 0.04
CTX/MESNA	0.38 ± 0.01^a	0.39 ± 0.04^a	022 ± 0.02^a	0.21 ± 0.02	1.12 ± 0.06^b	1.17 ± 0.10^b	3.19 ± 0.11	3.31 ± 0.07	0.08 ± 0.02^b	0.09 ± 0.03
CTX/extract	0.40 ± 0.03^a	0.41 ± 0.02^a	0.23 ± 0.03	0.24 ± 0.02^b	1.21 ± 0.12^b	1.26 ± 0.17^b	3.23 ± 0.14^b	3.26 ± 0.07^b	0.09 ± 0.02^b	0.10 ± 0.03
Extract only	0.41 ± 0.03	0.42 ± 0.01	0.22 ± 0.03	0.23 ± 0.01	1.16 ± 0.11	1.17 ± 0.15	3.28 ± 0.11	3.27 ± 0.08	0.08 ± 0.03	0.09 ± 0.02

Values are expressed as mean ± SD.

^ap < 0.01 and ^b p < 0.05: CTX/extract-co-treated or CTX/MESNA-co-treated values significantly different from that of CTX only.

Table 2. Effect of treatments on bone marrow cellularity and α-esterase activity in mice on Days 7 and 11.

	Bone marrow cellularity (cells/femur)		α-esterase activity (esterase^+ cells/4000 cells)
Treatment	Day 7 (×10^6)	Day 11 (×10^6)	Day 7	Day 11
Normal	17.18 ± 0.62)	18.04 ± 0.71	875.28 ± 12.28	971.24 ± 14.16
CTX control	9.80 ± 0.89	8.10 ± 1.04	652.23 ± 39.54	621.47 ± 33.28
CTX + MESNA	18.89 ± 0.47^a	19.15 ± 0.50^a	741.27 ± 34.27^a	765.31 ± 31.64^a
CTX + extract	19.53 ± 0.72^a	20.08 ± 0.64^a	775.58 ± 29.28^a	790.22 ± 33.25^a
Extract alone	20.14 ± 0.59^a	20.56 ± 0.73^a	935.26 ± 31.68^b	1012.24 ± 39.56^b

Values are expressed as mean ± SD.

^ap < 0.01: CTX/extract-co-treated or CTX/MESNA-co-treated values significantly different from that of CTX only.

^bp < 0.01: A. ferruginea extract alone value significantly different from that of naïve (normal) control.

Figure 3. Serum IL-2, IFNγ, GM-CSF, and TNFα after the respective treatments. Serum from the mice euthanized on Days 7 and 11 was assessed for each product by a specific ELISA. Values shown are mean ± SD. ^ap < 0.01 for CTX only versus CTX/extract- or CTX/MESNA-co-treated. ^cp < 0.01 for normal versus A. ferruginea extract alone.

Table 3. Effect of treatments on serum AST, ALT, and ALP in mice on Days 7 and 11.

	AST (U/L)		ALT (U/L)		ALP (U/L)
Treatment	Day 7	Day 11	Day 7	Day 11	Day 7	Day 11
Normal	40.12 ± 1.25	41.43 ± 1.67	35.25 ± 2.85	35.89 ± 2.26	82.26 ± 2.54	84.12 ± 1.92
CTX control	72.56 ± 4.57	76.39 ± 3.56	75.56 ± 3.58	78.59 ± 3.89	143.52 ± 5.59	145.64 ± 6.12
CTX + MESNA	52.56 ± 3.45^a	55.23 ± 2.86^a	43.87 ± 3.46^a	45.82 ± 3.96^a	85.26 ± 2.51^a	88.25 ± 3.54^a
CTX + extract	49.58 ± 3.96^a	51.23 ± 3.47^a	40.28 ± 2.56^a	41.78 ± 3.62^a	90.32 ± 3.58^a	92.51 ± 3.29^a
Extract alone	42.15 ± 1.56^c	43.89 ± 1.28^b	37.23 ± 2.12	38.25 ± 2.86	84.56 ± 2.17	85.28 ± 3.01

Values are expressed as mean ± SD.

^ap < 0.01: CTX/extract-co-treated or CTX/MESNA-co-treated values significantly different from that of CTX only.

^bp < 0.01, ^c p < 0.05: A. ferruginea extract alone value significantly different from that of naïve (normal) control.

Figure 4. Bladder LPO and GSH levels after the respective treatments. On Days 7 and 11, six mice from each group were euthanized and their bladders collected. An homogenate was prepared and used for measures of LPO and GSH. Values shown are mean ± SD. ^ap < 0.01 for CTX only versus CTX/extract- or CTX/MESNA-co-treated. ^cp < 0.01, ^dp < 0.05 for normal versus A. ferruginea extract alone.

Figure 5. Histology of small intestine due to the treatments. A representative micrograph from a mouse in each regimen is shown. (a) Naïve control showing normal mucosa. (b) CTX only (25 mg/kg BW) showing necrosis with damaged crypt architecture. (c) A. ferruginea extract (10 mg/kg BW) + CTX showing normal muscularis propria. (d) MESNA (25 mg/kg BW) + CTX with randomly damaged architecture. (e) Extract only showing normal architecture. All treatments were by IP injection of each treatment agent daily for 10 consecutive days. In the groups that received CTX or extract only, the volume of any missing component from the co-treatment groups was replaced by gum acacia-PBS injection so that all mice received the same volumes of injection each day. Magnification = 40×.

Figure 6. Histology of the urinary bladder due to treatments. A representative micrograph from a mouse in each regimen is shown. (a) Naïve control showing normal epithelium. (b) CTX only (25 mg/kg BW) showing damaged epithelium with edematous area. (c) A. ferruginea extract (10 mg/kg BW) + CTX showing normal lamina propria. (d) MESNA (25 mg/kg BW) + CTX showing normal mucosa. (e) Extract only showing normal architecture. All treatments were by IP injection of each treatment agent daily for 10 consecutive days. In all cases, the volume of any missing component for a given Group was replaced by phosphate buffer saline such that all mice received the same volume of injection each day. Magnification = 40×.

Table 4. Effect of treatments on small intestine and urinary bladder histology.

	Small intestine		Urinary bladder
Treatment	Day 7	Day 11	Day 7	Day 11
CTX control	Inflammation Noticeable coloration	Severe damage Red coloration Necrotic cells present	Inflammation Red coloration Hemorrhage	Severe inflammation Dark red coloration Severe hemorrhage Epithelial cells necrotized completely
CTX + MESNA	Slight inflammation Slight coloration	Still slight inflammation	Slight inflammation Slight coloration	No inflammation Normal architecture
CTX + extract	Slight inflammation Slight coloration	Still slight inflammation, but looked more normal	Slight inflammation Slight coloration	No inflammation Normal architecture
Extract alone	No inflammation	No inflammation	No inflammation and hemorrhage	No inflammation No hemorrhage

On Days 7 and 11, six animals from each group were euthanized and morphological examinations of the tissues for coloration, presence of hemorrhage, and extent of inflammation was performed in a blinded manner.