Relaxin prevents the development of severe acute pancreatitis

Table 1 Pancreatic and general biochemical assays in blood (mean ± SD)

Groups	Amylase	Lipase	Glucose	Urea	Creatinine	Calcium	Proteins	AST	ALT	Hto
	IU/L	IU/L	mg/dL	mg/dL	mg/dL	mg/dL	g/L	IU/L	IU/L	%
1 Basal	1103 ± 108	85 ± 18	92 ± 13	29 ± 3	0.35 ± 0.04	13.48 ± 0.39	85.5 ± 1.2	177 ± 25	88 ± 19	39 ± 5
2 Surgery control	1198 ± 114	98 ± 10	95 ± 10	38 ± 5	0.48 ± 0.11	13.22 ± 0.35	82.7 ± 0.8	205 ± 21	99 ± 21	31 ± 4
3 AP	7915 ± 159	5619 ± 194	338 ± 24	219 ± 19	1.82 ± 0.21	8.92 ± 0.16	61.1 ± 1.3	589 ± 26	295 ± 28	49 ± 9
4 AP+PBS	7921 ± 137	5628 ± 201	341 ± 19	217 ± 15	1.81 ± 0.16	8.94 ± 0.20	61.2 ± 2.2	591 ± 25	299 ± 28	49 ± 8
5 Rlx+LN	7154 ± 165	4982 ± 203	246 ± 23	183 ± 22	1.58 ± 0.22	9.37 ± 0.18	65.5 ± 1.8	544 ± 30	268 ± 19	47 ± 6
6 Rlx+MIF	7162 ± 171	4997 ± 207	249 ± 27	185 ± 24	1.60 ± 0.25	9.35 ± 0.14	65.1 ± 1.9	552 ± 28	270 ± 22	47 ± 8
7 Rlx+LN+MIF	7548 ± 175	5327 ± 214	262 ± 33	194 ± 21	1.69 ± 0.22	9.11 ± 0.21	62.4 ± 1.4	563 ± 34	281 ± 27	48 ± 5
8 Rlx 1h pre	6052 ± 169	3618 ± 155	190 ± 16	125 ± 11	1.13 ± 0.14	11.19 ± 0.22	74.5 ± 2.3	405 ± 24	196 ± 23	40 ± 4
9 Rlx 1h post	6427 ± 185	3910 ± 183	218 ± 15	146 ± 15	1.24 ± 0.19	10.91 ± 0.19	71.1 ± 1.9	452 ± 37	211 ± 27	45 ± 4
10 Rlx 1h pre+4h post	5589 ± 129	3161 ± 142	166 ± 18	97 ± 8	1.02 ± 0.17	11.98 ± 0.24	79.4 ± 1.6	382 ± 29	178 ± 23	42 ± 5
11 Rlx 4h post	6896 ± 192	4533 ± 178	235 ± 29	165 ± 19	1.39 ± 0.20	10.21 ± 0.19	69.9 ± 2.1	497 ± 33	235 ± 25	46 ± 5
12 Rlx 12h&1h pre+4h post	5535 ± 144	3153 ± 121	162 ± 22	95 ± 10	1.00 ± 0.15	12.03 ± 0.29	79.8 ± 1.4	395 ± 33	171 ± 25	43 ± 5

In relationship to Reference Basal-Group 1, BPDOE-CDLs AP-Group 3 presents a significant difference in all the assays evaluated P<0.05. The pancreatic and general biochemical profile significantly improved with relaxin treatment particularly for Groups 10 &12 that corresponded to administration times 12h and/or 1h pre and 4h post surgery P<0.05. Homogenous groups for amylase and lipase:1-2-(12-10)-8-9-11-(5-6-7)-(3-4), P<0.05. HTO: hematocrite, Rlx: relaxin, LN: L-NAME, MIF: mifepristone.

Table 2 Severity indicators assays in blood (mean ± SD)

Groups	Leukocytes	CRP	HSP 72	IL-6	IL-10	8-isoprostane
	nr/mL	mg/dL	pg/mL	pg/mL	pg/mL	pg/mL
1 Basal	6100 ± 224	0 ± 0	739 ± 32	below detection limit	below detection limit	0.11 ± 0.005
2 Surgery control	9856 ± 272	3.3 ± 1.02	1221 ± 21	207 ± 26	382 ± 59	0.13 ± 0.006
3 AP	26162 ± 258	30.9 ± 1.15	3396 ± 64	1962 ± 27	2216 ± 94	0.55 ± 0.011
4 AP+PBS	26145 ± 294	30.6 ± 1.21	3378 ± 56	1956 ± 32	2201 ± 85	0.56 ± 0.010
5 Rlx+LN	21223 ± 207	21.7 ± 1.24	2727 ± 48	1792 ± 28	2063 ± 90	0.49 ± 0.010
6 Rlx+MIF	21207 ± 236	21.4 ± 1.31	2765 ± 52	1784 ± 35	2052 ± 78	0.48 ± 0.009
7 Rlx+LN+MIF	23239 ± 273	27.3 ± 1.12	3023 ± 39	1849 ± 29	2113 ± 122	0.50 ± 0.008
8 Rlx 1h pre	17438 ± 266	13.9 ± 1.22	1924 ± 49	1473 ± 33	1695 ± 72	0.37 ± 0.010
9 Rlx 1h post	18776 ± 248	16.4 ± 1.52	2258 ± 42	1601 ± 25	1809 ± 87	0.42 ± 0.009
10 Rlx 1h pre+4h post	14527 ± 285	10.8 ± 1.44	1711 ± 39	1098 ± 32	1451 ± 68	0.31 ± 0.010
11 Rlx 4h post	19349 ± 225	19.5 ± 1.46	2506 ± 38	1689 ± 23	1975 ± 84	0.46 ± 0.008
12 Rlx 12h&1h pre+4h post	14542 ± 295	10.9 ± 1.27	1721 ± 43	1105 ± 28	1460 ± 76	0.32 ± 0.007

Severe AP was developed in groups 3 & 4. This severity was ameliorated by treatment with relaxin. In relationship to reference basal-group 1, BPDOE-CDLs AP in group 3 presented a significant difference in all the assays evaluated while treated groups 10 & 12 were the ones to exhibit the best prognosis with a significant improvement for all markers P<0.05.Homogenous groups for severity indicators :1-2-(12-10)-8-9-11-(5-6-7)-(3-4), P<0.05. Rlx: relaxin, LN: L-NAME, MIF: mifepristone.

Table 3 Assays in pancreatic and lung tissue (mean ± SD)

Groups	Panc. Amylase IU/mg DNA ds		Panc. Lipase IU/mg DNA ds		Panc. Proteins mg/mg DNA ds		Pancreatic MPO UMPO/mg DNA ds		Lung MPO UMPO/mg DNA ds	Panc. Wet Weight mg/100 g body wt
	H	B&T	H	B&T	H	B&T	H	B&T
1 Basal	995 ± 24	603 ± 27	98 ± 10	75 ± 11	185 ± 17	103 ± 12	0 ± 0	0 ± 0	0 ± 0	1310 ± 19
2 Surgery control	927 ± 19	576 ± 22	81 ± 11	66 ± 10	206 ± 19	122 ± 14	0 ± 0	0 ± 0	0 ± 0	1385 ± 24
3 AP	263 ± 17	181 ± 14	26 ± 9	16 ± 8	115 ± 10	57 ± 10	10.64 ± 0.39	7.15 ± 0.11	3.85 ± 0.13	2335 ± 38
4 AP + PBS	261 ± 20	185 ± 26	25 ± 9	15 ± 9	114 ± 11	58 ± 10	10.59 ± 0.31	7.22 ± 0.09	3.83 ± 0.12	2321 ± 43
5 Rlx + LN	392 ± 18	216 ± 21	34 ± 5	24 ± 6	129 ± 14	64 ± 8	8.12 ± 0.32	5.21 ± 0.12	2.93 ± 0.17	2159 ± 40
6 Rlx + MIF	389 ± 19	219 ± 23	35 ± 7	23 ± 5	130 ± 12	64 ± 13	8.09 ± 0.36	5.27 ± 0.15	2.96 ± 0.15	2142 ± 48
7 Rlx+ LN + MIF	315 ± 15	202 ± 12	32 ± 8	19 ± 4	121 ± 10	60 ± 8	9.01 ± 0.42	6.16 ± 0.10	3.10 ± 0.14	2198 ± 39
8 Rlx 1h pre	590 ± 29	342 ± 16	50 ± 12	37 ± 7	154 ± 18	81 ± 16	5.94 ± 0.22	3.68 ± 0.08	2.21 ± 0.08	1798 ± 33
9 Rlx 1h post	505 ± 33	307 ± 35	45 ± 10	32 ± 4	147 ± 20	70 ± 10	6.23 ± 0.25	3.95 ± 0.11	2.43 ± 0.09	1967 ± 39
10 Rlx 1h pre + 4h post	679 ± 31	387 ± 18	60 ± 9	43 ± 8	165 ± 23	90 ± 11	5.79 ± 0.19	3.59 ± 0.09	2.06 ± 0.10	1688 ± 29
11 Rlx 4h post	439 ± 22	273 ± 24	39 ± 9	27 ± 9	135 ± 15	66 ± 7	7.05 ± 0.28	4.45 ± 0.09	2.64 ± 0.14	2087 ± 42
12 Rlx 12h&1h pre + 4h post	683 ± 26	392 ± 17	59 ± 8	44 ± 9	167 ± 21	89 ± 10	5.78 ± 0.21	3.54 ± 0.03	2.07 ± 0.09	1693 ± 25

Pancreatic amylase and lipase decreased significantly in severe AP group 3, P<0.05. Treatment with relaxin induced a significant improvement with an increase in pancreatic enzymes and proteins P < 0.05, while there was a decrease in UMPO. This amelioration of the disease was even more remarkable for groups 10 & 12 indicating that treatment 12 and/or 1h pre and 4h post BPDOE-CDLs procedure had a protective role. The increase in pancreatic wet weight in groups with severe AP as a result of edema was significant P<0.01. Homogenous groups for pancreatic amylase-lipase and MPO: :1-2-(12-10)-8-9-11-(5-6-7)-(3-4), P<0.05; lung MPO: :1-2-(12-10)-8-9-11-(5-6-7)-(3-4), P<0.05. Rlx: relaxin, LN: L-NAME, MIF: mifepristone.

Table 4 Histopathological scores in pancreas and lung tissue (mean ± SD)

	Pancreatic Edema		Pancreatic emorrhage		Panc. Leuk. Infiltrate		Panc. Acinar Necrosis		Panc. Fat Necrosis		Lung Leuk
	H	B&T	H	B&T	H	B&T	H	B&T	H	B&T	Infiltrate
Groups
1 Basal	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
2 Surgery control	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
3 AP	1.31 ± 0.09	1.14 ± 0.08	3.58 ± 0.08	1.84 ± 0.06	3.61 ± 0.10	2.47 ± 0.08	3.87 ± 0.10	2.90 ± 0.08	3.98 ± 0.12	2.60 ± 0.08	3.65 ± 0.15
4 AP+PBS	1.33 ± 0.07	1.12 ± 0.10	3.55 ± 0.10	1.82 ± 0.07	3.64 ± 0.09	2.46 ± 0.11	3.83 ± 0.09	2.86 ± 0.12	3.95 ± 0.10	2.66 ± 0.11	3.61 ± 0.10
5 Rl x+LN	1.01 ± 0.06	0.73 ± 0.10	2.46 ± 0.10	1.08 ± 0.06	2.93 ± 0.14	1.53 ± 0.07	2.96 ± 0.10	2.03 ± 0.05	2.84 ± 0.11	1.57 ± 0.08	2.66 ± 0.10
6 Rlx+MIF	0.99 ± 0.10	0.75 ± 0.09	2.49 ± 0.06	1.27 ± 0.05	2.97 ± 0.11	1.58 ± 0.09	2.99 ± 0.09	2.00 ± 0.07	2.80 ± 0.07	1.61 ± 0.09	2.62 ± 0.07
7 Rlx+LN+MIF	1.26 ± 0.10	0.92 ± 0.08	2.97 ± 0.09	1.42 ± 0.05	3.23 ± 0.08	2.25 ± 0.06	3.35 ± 0.05	2.45 ± 0.08	3.00 ± 0.09	2.08 ± 0.10	3.04 ± 0.08
8 Rlx 1h pre	0.51 ± 0.08	0.42 ± 0.08	1.37 ± 0.07	0.59 ± 0.05	0.86 ± 0.08	0.45 ± 0.05	1.39 ± 0.11	0.85 ± 0.08	1.43 ± 0.10	0.75 ± 0.08	1.17 ± 0.09
9 Rlx 1h post	0.62 ± 0.07	0.50 ± 0.08	1.50 ± 0.08	0.71 ± 0.08	1.03 ± 0.03	0.62 ± 0.06	1.64 ± 0.07	1.01 ± 0.06	1.71 ± 0.08	0.98 ± 0.05	1.52 ± 0.10
10 Rlx 1h pre+4h post	0.42 ± 0.07	0.37 ± 0.06	1.26 ± 0.10	0.43 ± 0.07	0.72 ± 0.07	0.37 ± 0.09	0.92 ± 0.09	0.59 ± 0.05	1.19 ± 0.09	0.63 ± 0.09	0.80 ± 0.08
11 Rlx 4h post	0.77 ± 0.08	0.59 ± 0.07	1.97 ± 0.05	0.89 ± 0.09	1.67 ± 0.05	0.89 ± 0.08	2.28 ± 0.13	1.45 ± 0.08	2.23 ± 0.09	1.19 ± 0.07	2.11 ± 0.05
12 Rlx 12h&1h pre+4h post	0.45 ± 0.05	0.36 ± 0.05	1.24 ± 0.08	0.45 ± 0.04	0.70 ± 0.05	0.38 ± 0.06	0.90 ± 0.05	0.60 ± 0.05	1.20 ± 0.05	0.61 ± 0.07	0.83 ± 0.06

Groups 3&4 that developed severe AP presented as main features leukocyte infiltrate, hemorrhage and acinar and fat necrosis and the histopathological scores were higher. Groups 10 & 12 that received relaxin 12 and/or 1h pre and 4h post surgery developed a mild AP and exhibited the lowest scores being the main feature edema. Homogenous groups for pancreatic edema, hemorrhage, acinar and fat necrosis and pancreatic and lung leukocyte infiltrate: :1-2-(12-10)-8-9-11-(5-6-7)-(3-4), P < 0.05. Rlx: relaxin, LN: L-NAME, MIF: mifepristone.

Table 5 Order of groups according to the increase in the severity degree

GROUP NR.	GROUP DESCRIPTION
1	Basal Reference Control
2	Surgery Control
12	Relaxin administered 12h & 1h pre- BPDOE-CDLs and 4h post-BPDOE-CDLs
10	Relaxin administered 1h pre- BPDOE-CDLs and 4h post-BPDOE-CDLs
8	Relaxin administered 1h pre-BPDOE-CDLs
9	Relaxin administeres 1h post-BPDOE-CDLs
11	Relaxin administered 4h post-BPDOE-CDLs
5	Relaxin and L-NAME administered 1h pre- BPDOE-CDLs and 4h post-BPDOE-CDLs
6	Relaxin and Mifepristone administered 1h pre- BPDOE-CDLs and 4h post-BPDOE-CDLs
7	Relaxin, L-NAME & Mifepristone administered 1h pre- BPDOE-CDLs and 4h post-BPDOE-CDLs
3	BPDOE-CDLs AP model
4	Vehicle (PBS) control

The BPDOE-CDLs model induces a severe AP (group 3).Prophylactic treatment with relaxin, as shown for groups10&12, markedly reduces the severity of the pathology, being still useful although in a lesser degree even after AP was triggered.